Ngal for reduction and amelioration of ischemic and nephrotoxic injuries

ABSTRACT

Use of neutrophil gelatinase-associated lipocalin (NGAL) as a therapeutic and in a method of treating, reducing, or ameliorating an injury selected from an ischemic injury, an ischemic-reperfusion injury, and a toxin-induced injury, to an organ in a patient. The invention includes administering to the patient NGAL in an amount effective to treat, reduce or ameliorate ischemic, ischemic-reperfusion, or toxin-induced injury to the organ, such as the kidney. A siderophore can be co-administered with the NGAL. The invention also relates to administering a sideophore to enhance a response to secretion of NGAL following an ischemic or toxin-induced injury to an organ in a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/123,364 filed May 6, 2005 which claims the benefit of U.S.Provisional Application Nos. 60/586,645, filed May 6, 2004 and60/615,566, filed Oct. 1, 2004.

INTERESTS

This invention was made with Government support awarded by the NationalInstitute of Health (NIH)/National Institute of Diabetes and Digestiveand Kidney Diseases, under Grant Nos. DK53289, DK52612, and DK070163.The Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods of reducing or amelioratingischemic and nephrotoxic injury in organs. The invention further relatesto the treatment of patients suffering from various diseases that areassociated with ischemic and toxin-induced injury or insult. Theinvention further relates to the treatment of patients suffering fromacute or chronic kidney injury.

BACKGROUND OF THE INVENTION

A decrease in oxygen flow to an organ, called ischemia, triggers acomplex series of events that affect the structure and function ofvirtually every organelle and subcellular system of the affected cells.Ischemia-reperfusion (resumption of blood flow) injury leads to theproduction of excessive amounts of reactive oxygen species (ROS) andreactive nitrogen species (RNS), thus causing oxidative stress whichresults in a series of events such as alterations in mitochondrialoxidative phosphorylation, depletion of ATP, an increase inintracellular calcium and activation of protein kinases, phosphatases,proteases, lipases and nucleases leading to loss of cellularfunction/integrity. It has been shown that the inflammatory responseinduced by ischemia followed by reperfusion is largely responsible fortissue and organ damage.

Ischemic-reperfusion injury is a serious problem in organtransplantation because the harvested organ is removed from the body,isolated from a blood source, and thus deprived of oxygen and nutrientsfor an extended period of time. A critical problem to be addressed inpresent-day kidney transplantation procedures is the relatively highincidence of delayed graft function (DGF) due to acute tubular necrosis(ATN) following surgery. Illustratively, DGF affects about 20-35% ofkidney transplants in many transplant centers and is the most commoncomplication of the immediate post-operative period in renaltransplantation. Although the incidence and definition of DGF vary amongtransplant centers, the consequences most frequently involve a prolongedhospital stay, additional invasive procedures and additional costs tothe patient and health-care system. Delay in graft function not onlyaffects the individual patient, it also impacts the infrastructure fororgan procurement and sharing as a consequence of the drain it places onthe available organ supply. DGF also increases the risk of early acuterejection episodes and increases early graft loss from chronicrejection.

With current preservation methods, cold ischemia resulting from organpreservation has been identified as a major risk factor in causing DGFafter transplant. For kidneys, cold ischemia times in excess of 24 hoursare associated with a significantly increased risk of DGF. In themid-1960's, cold preservation of kidneys was effectively achieved byusing machine perfusion and a solution derived from cryoprecipitatedplasma. Thereafter, by simple cold-storage methods were introduced andinvolved the use of a cold crystalloid solution. Since the earlysuccesses with kidneys, preservation solutions have evolved intoentirely synthetic defined media designed to prevent cold ischemicinjury by suppression of cell swelling and provision of metabolicsupport. An early synthetic solution, a lactobionate-based solution (UW)from the University of Wisconsin has been used for both pancreas andliver. With the advent of synthetic and serum-free preservationformulations, the quality and duration of feasible organ preservationhave improved. Despite this, however, clinical data on DGF in kidneysand other problems involving renal cell structure and morphology clearlydemonstrate that such solutions are not completely successful inpreventing ischemic injury or insult.

In addition, acute renal failure (ARF) secondary to ischemic ornephrotoxic injury also remains a common and potentially devastatingproblem in clinical nephrology, with a persistently high rate ofmortality despite significant advances in supportive care. Over severaldecades, a number of studies have illuminated the roles of persistentvasoconstriction, tubular obstruction, cellular structural and metabolicalterations, and the inflammatory response in the pathogenesis of ARF.Treatments and remedies for ARF have been hampered by the multifacetedresponse of the kidney to ischemia, as well as a lack of early markersfor ARF. Recent advances in cellular and molecular biology of ischemicand nephrotoxic renal injury have shown that proximal tubule cellsundergo a complex temporal sequence of events, including loss of cellpolarity, cell death due to apoptosis and necrosis, de-differentiationand proliferation of viable cells, and re-establishment of theepithelial phenotype.

As a result of ischemic or nephrotoxic damage, cells may die through twodifferent processes. Apoptosis or programmed cell death (“cell suicide”)is a physiological mechanism for removing senescent, damaged or abnormalcells that affects individual cells. Apoptosis is initiated by anendonuclease and is characterized by DNA fragmentation into multiples of180-200 base pairs. Apoptotic cells are ingested by macrophages orneighboring cells without release of proteolytic enzymes or toxic oxygenspecies and the process is not accompanied by inflammation. By contrast,necrosis (“cell murder”) is a pathological process that affectspopulations of cells and results in focal tissue destruction,inflammation and often serious systemic consequences. Apoptotic celldeath has now been shown to play an important role in an increasingarray of kidney diseases, including ischemia, ischemia-reperfusion,nephrotoxins, polycystic kidney disease, obstruction, and glomerulardiseases. Down-regulation of apoptosis therefore offers a unique andpowerful therapeutic approach to the amelioration of several acute andchronic kidney injuries.

An individual is considered to have acute renal failure when thepatient's serum creatinine value either (1) increased by at least 0.5mg/dL when the baseline serum creatinine level was less than 2.0 mg/dL;(2) increased by at least 1.5 mg/dL when the baseline serum creatininelevel was greater than or equal to 2.0 mg/dL; or (3) increased by atleast 0.5 mg/dL, regardless of the baseline serum creatinine level, as aconsequence of exposure to radiographic agents.

cDNA microarray techniques have allowed the identification of neutrophilgelatinase-associated lipocalin (NGAL) as a highly induced transcript inthe kidney early after ischemic and nephrotoxic injury. The role of NGALin the kidney has yet to be elucidated. NGAL is a member of thelipocalin family of proteins and is characterized as a secreted 25 kDaglycoprotein found in granules of human neutrophils. (Kjeldsen et al,1993, J. Biol. Chem. 268:10425-10432). Lipocalins, which are able tobind small lipophilic substances, share a common three-dimensionaln-barrel structure which functions, in at least some lipocalins, to bindlipophilic ligands, e.g., steroid, bilin, retinoid, or other lipid.Murine forms of NGAL (homologs) from mice and rats are known. In mice,NGAL has been designated as NGAL, 24p3 protein, SIP24, P25, lipocalin 2,and uterocalin. NGAL in rats is known as NGAL or alpha 2-microglobulin.A full-length cDNA encoding human NGAL protein has been cloned andsequenced. The human NGAL gene, which includes seven exons and sixintrons, has also been cloned and sequenced, and its expression invarious tissues has been analyzed. The human NGAL gene encodes apolypeptide of 197 amino acids, with a 19- or 20-amino acid signalsequence, and a mature NGAL polypeptide containing 178 amino acids. Themotifs Gly-X-Trp (amino acids 48-50 in mature human NGAL) andThr-Asp/Asn-Tyr (amino acids 132-134 in mature human NGAL) are presentin all known lipocalins. On the basis of X-ray crystallography, it hasbeen suggested that these motifs are important in the tertiary β-barrelstructure shared among the lipocalins. The cysteine residues 95 and 194in the human NGAL sequence are conserved, and have been reported to forman intramolecular disulfide bridge. Human NGAL contains a singleN-glycosylation site (an asparagine residue) at position 65 of themature amino acid sequence (approximately position 84 or 85 of thepre-NGAL polypeptide).

A mechanism that may underlie ATN is mis-localized iron. Unbound ironcan catalyze the conversion of H₂O₂ to OH and OH⁻ (the Haber-Weissreaction) or form reactive ferryl or perferryl species. These ionsmutagenize many types of molecules including lipids, nucleotides and theDNA backbone. Catalytic iron, released from free hemoglobin andmyoglobin into urine or blood, and peroxidized lipids have beendocumented in many forms of acute renal failure, including chemotherapy,ischemia-reperfusion, transplant ischemia, and in proteinuria-mediatedtubular damage. Preloading animals with iron worsens the disease, andconversely chelating iron with deferoxamine or bacterial siderophoresblunts the damage. Iron-catalyzed damage is thought to be one of theearliest events in kidney dysfunction and is likely to be important inother organs, including the heart and the liver (See Mori et al:Endocytic delivery of lipocalin-siderophore-iron complex rescues thekidney from ischemia-reperfusion injury. J Clin Invest 115:610-621,2005).

Cells acquire iron from carrier proteins (such as transferrin) and bycell surface iron transporters (such as divalent metal transporter I).Intracellular iron is controlled by the actions of the iron responsiveproteins (such as IRP1, IRP2), the ferritin complex and heme oxygenaseI. Because IRPs are modulated by hypoxia, oxidative stress, andphosphorylation, changes in their activity may play an important role inischemic disease, by regulating formation of ferritin complexes, whichprotect cells from iron mediated damage. Ferritin is aniron-phosphorous-protein complex, comprising approximately 23% iron,formed in the intestinal mucosa. Ferritin is the storage form of iron intissues such as liver, spleen, and bone marrow. Hemoglobin and myoglobinmolecules in blood and muscle, respectively, require iron-binding tocatalyze transfer of oxygen to cells. However, few other aspects of irontrafficking, storage or metabolism are known in ischemic cells or inother types of tissue damage, despite the primacy of catalytic iron intheir pathogenesis.

Despite the many pathways of producing ATN, a number of investigatorshave discovered general underlying mechanisms of proximal tubule celldamage. These have included the release of cytokines. One idea is thatischemic cells and tubular toxins such as free myoglobin and hemoglobinproduce high concentrations of iron locally in the nephron. It isthought that this iron is catalytically active, and produces oxygenradicals. Evidence that iron catalyzed cell damage is pathogenic andleads to proximal tubular dysfunction includes the finding that tubulardamage is blunted by infusions of iron chelators. Additional support forthe idea that iron is central to the mechanism of organ dysfunctionafter ischemia comes from experiments that used iron free-bacteriallyderived, iron chelators, called siderophores, to blunt the effects ofischemia-reperfusion injury in an in vitro model of cardiac ischemia.Each of these general mechanisms is thought to be the principlepathogenic event during different stages of ATN. Iron catalyzed damageis thought to be one of the earliest events in kidney dysfunction.

It is currently unknown how the proximal tubule captures NGAL. Indeed anunambiguous identification of receptors for most lipocalins is stilllacking. Perhaps megalin, which is necessary for reclamation of RBP, isalso the NGAL receptor (Christensen et al., 1999, J Am Soc Nephrol.10(4) 685-95). In fact, knockout of megalin leads to the appearance ofNGAL in the urine, but these animals were also, unexpectedly, found tohave much higher levels of NGAL message (Hilpert et al., 2002, KidneyInt 62(5)1672-81), suggesting that urinary NGAL might have derived fromlocal synthesis rather than a failure to capture the filtered load.Despite this ambiguity, NGAL is similar to other lipocalins, such as RBPand α-2u globulin lipocalin (see Borghoff et al., 1990, Annu RevPharmacol Toxicol. 30:349-67), which enter the cell by a megalin pathwayand traffic to lysosomes for degradation. These data contrast with thetrafficking of NGAL in cell lines that do not express megalin (such asembryonic kidney cells) and where the protein escapes degradation (seeYang et al., 2002, Mol. Cell. 10(5):1045-56). Similarly, transferrin isalso degraded after delivery to lysosomes by a megalin-cubulin basedpathway in the proximal tubule (see Kozyraki et al., 2001, Proc. Natl.Acad Sci USA 98(22)12491-6), whereas it usually recycles in cell lines.Hence it is reasonable to propose that after filtration, NGAL iscaptured by megalin and degraded by the proximal tubule and is notrecycled. This hypothesis is supported by the observation that fulllength NGAL does not reappear in the blood at delayed time pointspost-injection.

There remains a need for compositions and methods suitable forpreventing, reducing, or ameliorating ischemic injury, e.g., coldischemic injury, in organs such as the kidney. Such compositions wouldbe useful both in treating a patient's original organs, as well asorgans used for transplantation. Also needed are new biomarkers that canbe used to detect toxic damage to cells, for example nephrotoxicity, inpatients following drug administration. New and improved methods oftreating and reducing ischemic-reperfusion injury to tissues and organscaused by organ transplantation, and of treating and reducing structuraland metabolic alterations of organ cells, are clearly useful andimportant to practitioners and patients alike.

SUMMARY OF THE INVENTION

The present invention relates to neutrophil gelatinase-associatedlipocalin (NGAL) and its use in compositions and methods for treating,reducing, ameliorating, or preventing a condition, injury or disease,typically selected from an ischemic, an ischemia-reperfusion, or atoxin-induced injury in an organ. The invention also relates to a methodof administering to a patient or subject NGAL in an amount effective totreat, reduce, ameliorate or prevent the condition, injury or disease.The injury can include a renal injury associated with conditions,treatments, therapies, or diseases that predispose a patient to ischemicrenal injury, a renal tubule injury, or necrosis/apoptosis. The injurycan include acute (including but not limited to shock, stroke, sepsis,trauma, infection, inflammation) or chronic (including but not limitedto hypertension, diabetes, heart failure, lupus, infections,inflammations) kidney conditions.

The present invention also provides a method of ameliorating reductionin kidney NGALfunction induced by ischemia-reperfusion injury in apatient by administering NGAL to the patient in an amount effective toameliorate the reduction of kidney function. In accordance with thisaspect, NGAL administration reduces high levels of serum or plasmacreatinine following ischemia-reperfusion injury. The amount of NGALadministered is effective to prevent or ameliorate cell death.

The invention further provides a method of enhancing renalre-epithelialization or tubular cell proliferation following anischemic, ischemic-reperfusion, or toxin-induced injury, and in acute orchronic kidney disease, by administering NGAL to a patient in an amounteffective to enhance renal re-epithelialization or effect proliferationof tubular cells.

An embodiment of the invention includes the use of NGAL in a method oftreating, reducing, preventing or ameliorating acute renal failure(ARF). NGAL has been demonstrated to enhance tubule cell proliferationand reduce or ameliorate tubule cell apoptosis. The methods disclosedcan ameliorate the reduction in kidney function in a patient that isinduced by an ischemia-reperfusion or toxin-induced injury, and canfurther be used for treating, reducing, ameliorating or preventing acuterenal failure secondary to ischemic injury in the patient.

The invention further provides the use of NGAL in a method for reducingand/or treating delayed graft function (DGF) of a transplanted organ ina patient. In some aspects, DGF is caused by acute tubular necrosis(ATN).

The invention also provides the use of NGAL in a method fortransplanting and grafting of an organ into a patient. The use of themethod can reduce or ameliorate organ graft or transplant loss or acuterejection by introducing NGAL into one or more of (i) the organ or (ii)a donor thereof, in an amount effective to reduce or ameliorate loss oracute rejection of the organ graft or transplant. In various aspects,the organ is selected from kidney, liver, heart, brain, lung, stomach,intestine, colon, pancreas, blood vessels, bladder, cervix, skin, or aportion or section thereof. In a particular aspect, the organ resides ina cadaverous or living organ donor. In this situation, NGAL can beadministered to the patient before, during and/or after the organ istransplanted or grafted into the patient. The organ can be a cadaverousorgan, and in those instances in which the organ is obtained from acadaverous donor, NGAL can be administered to either the cadaver or theextracted organ to prevent injury, insult, or failure of the organfollowing transplantation. The organ can be a living organ donation, andin those instances NGAL can be administered to the extracted organ toprevent injury, insult, or failure of the organ followingtransplantation. The organ can be a kidney, liver, heart, brain, lung,stomach, pancreas, blood vessels, bladder, cervix, skin, or a portion orsection thereof. In a particular aspect of the present invention, theorgan is a kidney.

The present invention also provides the use of NGAL in association withcadaveric and living donor renal transplantation, where oxidant-mediatedapoptosis is an important contributor to tubule cell death. In additionto the usual complications of acute renal failure (ARF),ischemia-reperfusion injury in the transplanted kidney is known toresult in delayed graft function (DGF), which significantly increasesthe risk of graft loss and acute rejection. As described herein, NGAL isemployed in methods for reducing or ameliorating the adverse ischemiceffects associated with organ transplantation. In one embodiment, NGALcan be added to an organ preservation solution, such as is used duringcold storage of transplant organs, to ameliorate the DGF that ischaracteristic of cadaveric kidney transplantation. In accordance withthis method, NGAL is at least partially effective even when administeredafter the ischemic insult. Advantageously, the method affords needed andnovel therapeutic treatments of established ischemic conditions, such asARF, which is an existing, clinically-relevant, adverse event that iscommonly associated with a dismal prognosis for the patient. The methodof retarding or ameliorating DGF associated with ischemic injury in anorgan or graft transplant in a patient includes introducing an amount ofNGAL into, or contacting NGAL with, (i) a transplanted organ or graft;(ii) a donor of a transplanted organ; or (iii) both (i) and (ii), in anamount effective to retard or ameliorate DGF, or the loss or acuterejection of the transplanted or grafted organ. Illustratively, andwithout limitation, the organ or graft for transplant is selected fromkidney, liver, heart, brain, lung, stomach, intestine, colon, pancreas,blood vessels, bladder, cervix, skin, or a portion or section thereof.In a particular aspect, the organ is a kidney. More particularly, thekidney transplanted is a cadaveric or living donor kidney. Further, NGALis a component of the organ preservation solution, e.g., anNGAL-containing organ preservation solution is used during cold storageof the organ transplant.

The present invention further provides a method of reducing,ameliorating, preventing or protecting a patient from renal injury thatis associated with conditions, treatments, therapies, or diseases thatcan predispose a patient to ischemic renal injury. The method comprisesadministering to the patient an amount of NGAL effective to reduce,ameliorate, prevent or protect the patient from renal injury associatedwith the patient's condition, treatment, therapy, or disease.Illustrative conditions, treatments, or therapies include withoutlimitation, contrast agent treatment, antibody treatment, antibiotictreatment, organ transplant, kidney transplant, cadaveric kidneytransplant, cardiac treatment, cardiac treatment after surgery, orcentral nervous system treatment. Illustrative diseases according tothis aspect include, without limitation, infection, bacterial infection,acute kidney disease, chronic kidney disease, ischemic-reperfusioninjury, shock, trauma, sepsis, stroke, cardiac reperfusion injury,cardiopulmonary bypass, open heart surgery, and abdominal surgery.

The invention also provides a method of treating, reducing orameliorating renal tubule injury or necrosis/apoptosis in a patient,which comprises administering a therapeutically effective amount of NGALto the patient. According to this aspect, the patient can be affectedwith acute kidney disease, chronic kidney disease, ischemic-reperfusioninjury, organ transplant, toxin-induced injury, ischemia, kidneytransplant, shock, trauma, sepsis, stroke, cardiac reperfusion injury,renal tubule injury following cardiopulmonary bypass, renal tubuleinjury following open heart surgery, renal tubule injury followingabdominal surgery, infection, antibiotic treatment, antibody treatment,or contrast agent treatment, for example. In the method, NGAL canfunction to enhance proliferation of renal tubule cells, since NGAL alsodirectly targets renal proximal tubule cells, resulting in reduction oramelioration of renal tubule injury or necrosis/apoptosis.

The present invention also provides a method of treating, reducing, orameliorating a toxin-induced injury, including a nephrotoxic injury, toan organ in a patient by administering NGAL to the patient in an amounteffective to treat, reduce or ameliorate the toxin-induced injury to theorgan. In another aspect, the method of reducing or ameliorating atoxin-induced injury includes co-administering both NGAL and atherapeutic compound that is toxic to the patient, the NGAL beingadministered in an amount effective to reduce or ameliorate the toxiceffect of the therapeutic on the organ.

The present invention also provides a method of treating, reducing, orameliorating other acute (including but not limited to shock, stroke,sepsis, trauma, infection, inflammation) and chronic (including but notlimited to hypertension, diabetes, heart failure, lupus, infections,inflammations) kidney injuries.

In the various methods of the present invention disclosed herein, NGALcan be administered in conjunction with one or more therapeutic agents,such as, for example, vasodilators or oxygen supplying agents. Inaddition, NGAL can be administered in a physiologically acceptablecomposition comprising a carrier, diluent, or excipient by variousroutes of administration, including, without limitation, intravenous orparenteral routes. Further, NGAL can be administered prior to, during,or following ischemia or nephrotoxicity, organ transplant or grafting,or renal tubule insult, damage, or injury, as described herein.

The present invention also provides a method of evaluating a therapeuticfor its potential to induce nephrotoxicity by (a) administering a testsubstance to cause a nephrotoxic injury a mammalian model, such as amouse; and (b) determining the presence of neutrophilgelatinase-associated lipocalin (NGAL) in a urine or plasma sample ofthe mammalian model following the administration of the test substance,as an indication that the substance can induce kidney damage. In aparticular aspect of this method, NGAL is detected in the urine orplasma within about three hours following administration of the testsubstance.

The present invention also relates to a composition for use in thetreating, reducing, ameliorating, or preventing an injury to an organ ina mammal, comprising a therapeutically-effective amount of NGAL, or aderivative or analog thereof. The composition can further a siderophore,typically in a 1:1 molar ratio, including a complex of the NGAL with thesiderophore. The composition can be used in any of the methods disclosedherein.

The present invention also relates to the use of siderophores inassociation with NGAL in a composition and a method for treating,reducing, ameliorating, or preventing an ischemic or toxin-inducedcondition and disease, including an ischemic, an ischemia-reperfusion,or a toxin-induced injury in an organ.

In one embodiment of the invention, NGAL and a siderophore areadministered as a pharmaceutical composition in an amount effective toenhance the treatment, reduction, amelioration or prevention of anischemic, an ischemia-reperfusion, or a toxin-induced injury in anorgan.

The present invention also relates to a pharmaceutical composition thatcomprises a siderophore, for use in administration to patients toenhance treatment prevention, amelioration, and reduction of injury inan organ by endogenous NGAL. In another embodiment, a method is providedfor co-administering, typically as a complexed compound, of NGAL and asiderophore. In another embodiment, a siderophore is administered to apatient in an amount effective to enhance the renal re-epithelializationor positively affect the proliferation of tubular cells initiated byendogenous NGAL secretion.

In particular, the invention provides a method of enhancing thereduction or amelioration of delayed graft function (DGF) and organ orgraft transplant rejection in a patient by endogenous NGAL, comprisingthe step of introducing a siderophore into (i) a transplanted organ orgraft; (ii) a donor of a transplanted organ; or (iii) both (i) and (ii),in an amount effective to enhance the reduction or amelioration of DGF,or the loss or acute rejection of the transplanted or grafted organ. Themethod includes administering a siderophore in an amount effective totreat, reduce, ameliorate or prevent the injury to the organ.

In an embodiment of the invention, NGAL:siderophore complexes can beadded to an organ preservation solution, such as is used during coldstorage of transplant organs, to ameliorate the DGF that ischaracteristic of cadaveric kidney transplantation. In yet anotherembodiment, siderophores alone in a buffer solution can be added to anorgan preservation solution, such as is used during cold storage oftransplant organs, to ameliorate the DGF that is characteristic ofcadaveric kidney transplantation.

The invention further provides a method for manipulating cellular andextracellular iron in an ischemic or toxin-damaged organ byadministering NGAL, or a derivative or analog thereof. This method canalso include administering an iron-binding chemical, a co-factor for aniron-binding chemical, or a siderophore in an amount effective to treat,reduce, ameliorate or further prevent organ damage by ischemia ortoxins. Typically the ischemic or toxin-damaged organ is a kidney.

In another embodiment, the present invention provides a therapeutic kitcomprising a first container for containing a therapeutically-effectiveamount of NGAL. The first container can include at least one vial, atleast one test tube, at least one flask, and at least one bottle. Thekit can also include a second container into which at least onecomposition can be placed, and a means for securing the containerstogether for commercial sale. The kit can also include a third containerfor containing a sterile, pharmaceutically acceptable buffer or otherdiluent. The first container can be a syringe. The means for securingthe containers can be an injection or blow-molded plastic container intowhich the containers are retained. Alternatively, the vials can beprepared in such a way as to permit direct introduction of thecomposition into an intravenous drug delivery system. Instructions foruse are also typically included.

Further aspects, features and advantages of the present invention willbe better appreciated upon a reading of the detailed description of theinvention when considered in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Coomassie Blue (CB) and enhanced chemiluminescence (ECL,with polyclonal NGAL antibody) analysis of defined quantities (as shown)of recombinant purified NGAL.

FIG. 2 shows immunofluorescent staining of kidneys from controlnon-ischemic animals one hour after injection, or ischemic kidneys oneor three hours after either injection, using polyclonal NGAL antibody.

FIG. 3 shows Western blot for NGAL detection in urine samples fromnon-ischemic (NI) and ischemic (I) animals within 1 hour ofadministration of NGAL.

FIG. 4 shows sections stained with hematoxylineosin of kidneys fromcontrol non-ischemic mice, saline pre-treated ischemic mice, or ischemicmice treated with NGAL one hour before, during, or one hour afterischemia.

FIGS. 5A, 5B and 5C show scoring of histological sections of kidneysfrom ischemic mice that were saline pre-treated or treated with NGAL onehour before, during, or one hour after ischemia. The sections wereanalyzed and scored for tubule dilatation (FIG. 5A), tubule casts (FIG.5B), and tubule cell necrosis (FIG. 5C) using an arbitrary scale of 0 to4.

FIG. 6 shows serum creatinine measured in non-ischemic (Non Isch)control mice, or 24 hours following ischemia in mice pre-treated withsaline (Pre Sal) or treated with NGAL one hour before (Pre NGAL), during(Dur NGAL) or one hour after (PostNGAL) ischemic injury.

FIG. 7 shows results of TUNEL staining of representative sections fromnon-ischemic control mice, or 24 hours following ischemia in micepre-treated with saline or treated with NGAL one hour before, during, orone hour after ischemic injury. Arrows point to the condensed,fragmented, intensely staining nuclei characteristic of apoptosis in lowand high power magnifications, compared to staining with proliferatingcell nuclear antigen (PCNA).

FIG. 8 shows quantitation of apoptosis (upper panel) and proliferation(center panel) in kidneys from non-ischemic control mice, or 24 hoursfollowing ischemia in mice pre-treated with saline or treated with NGALone hour before, during, or one hour after ischemic injury.

FIG. 9 shows a ratio of proliferation:apoptosis calculated in kidneysfrom non-ischemic control mice, or 24 hours following ischemia in micepretreated with saline or treated with NGAL one hour before, during, orone hour after ischemic injury.

FIG. 10 shows binding of ⁵⁵Fe detected in urine alone, a urine and NGALmixture (urine+Ngal), a urine and NGAL:siderophore mixture(urine+Ngal:Si), plasma alone, buffer alone, buffer and NGAL mixture(buffer+Ngal), a buffer and NGAL:siderophore mixture(buffer+Ngal:Sid(1)), a second buffer and NGAL:siderophore mixture(buffer+Ngal:Sid(2)), and a third buffer and NGAL:siderophore mixture inwhich the siderophore was saturated with iron (buffer+Ngal:Sid:Fe).

FIG. 11A shows immunoblots of NGAL protein in human urine samples fromhealthy subjects (Normal), or subjects with Acute Tubular Necrosis (ATN)or chronic renal failure (CRF), or from subjects with liver cirrhosis,hemochromatosis, or pancreatic carcinoma but lacking a renal diagnosis(Others). Monoclonal anti-human NGAL (Mo) and polyclonal anti-mouse NGAL(Po) antibodies recognizes recombinant and native human NGAL and NGALStandards.

FIG. 11B shows immunoblots of NGAL protein in human serum samples fromhealthy subjects (Normal), or subjects with Acute Tubular Necrosis (ATN)or chronic renal failure (CRF), or from subjects with liver cirrhosis,hemochromatosis, or pancreatic carcinoma but lacking a renal diagnosis(Others), compared to NGAL Standards.

FIG. 11C shows a quantitative comparison of NGAL protein levels in urinefrom healthy subjects (Normal), or subjects with Acute Tubular Necrosis(ATN) or chronic renal failure (CRF). ATN is further subdivided intonon-sepsis and sepsis groups.

FIG. 11D shows a quantitative comparison of NGAL protein levels in serumfrom healthy subjects (Normal), or subjects with Acute Tubular Necrosis(ATN) or chronic renal failure (CRF). ATN is further subdivided intonon-sepsis and sepsis groups.

FIG. 11E shows an immunoblot of NGAL in urine from ATN-injured andSham-treated control mice, along with standards of 50, 5, and 1 ng NGALprotein.

FIG. 12A shows immunohistochemical staining for NGAL in a healthy humankidney (Normal).

FIG. 12B shows immunohistochemical staining for NGAL in a healthy humankidney.

FIG. 12C shows immunohistochemical staining for NGAL in a healthy humankidney.

FIG. 12D shows increased immunohistochemical staining for NGAL in ahuman kidney with ischemic ATN caused by sepsis (Ischemic ATN).

FIG. 12E shows increased immunohistochemical staining for NGAL in ahuman kidney with ischemic ATN caused by hypovolemia due to vomiting anddiarrhea.

FIG. 12F shows increased immunohistochemical staining for NGAL in ahuman kidney with ischemic ATN caused by heart failure.

FIG. 12G shows increased immunohistochemical staining for NGAL in ahuman kidney with toxic ATN caused by nephrotoxicity due tobisphosphonate (Toxic ATN).

FIG. 12H shows increased immunohistochemical staining for NGAL in ahuman kidney with toxic ATN caused by nephrotoxicity due tocephalosporin toxicity.

FIG. 12I shows increased immunohistochemical staining for NGAL in ahuman kidney with toxic ATN caused by nephrotoxicity due tohemoglobinuria.

FIG. 12J shows immunohistochemical staining for NGAL in a human kidneywith glomerular disease, with NGAL weakly expressed in cresents.

FIG. 12K shows immunohistochemical staining for NGAL in a human kidneywith glomerular disease, with NGAL weakly expressed in the proximaltubules of nephrotics.

FIG. 13A shows histological sections of sham-treated (Control), ischemic(ATN), and NGAL-treated ATN-injured (ATN+Ngal) kidneys. Loss of tubularnuclei is observed in ATN but not control or ATN+Ngal sections (upperpanels), as well as cortical (center panels) and medullary (lowerpanels) intratubular casts. NGAL pretreatment resulted in preservationof cortical tubules, but residual cortical-medullary casts.

FIG. 13B shows histological sections of ischemic (ATN), and NGAL-treatedATN-injured (ATN+Ngal) kidneys, with PAS staining highlighting theluminal casts and the rescue of cortical tubules by pre-treatment withNGAL.

FIG. 13C shows Jablonski scoring of sham-treated (Sham), ischemic (ATN),and NGAL-treated ATN-injured (ATN+Ngal) kidneys to demonstrate rescue ofthe ischemic cortex by NGAL.

FIG. 14A shows N-cadherin staining in kidney sections is nearlyabolished by ischemia reperfusion (ATN), but is rescued when NGAL isadministered (ATN+Ngal).

FIG. 14B shows full length N-cadherin protein levels are rescued by NGALtreatment (ATN+Ngal), compared to ischemic (ATN), as indicated by theN-cadherin fragments (arrow) in ischemia-reperfusion and sham-treatedanimals, but their suppression in NGAL-treated animals. GAPDH is theloading control.

FIG. 14C shows tubules with TUNEL-positive apoptotic cells(fluorescence) in ischemic-reperfused mice (I/R) reduced by pretreatmentwith NGAL (I/R+Ngal). Toprol is the nuclear counterstain.

FIG. 14D shows a quantitative analysis of the percentage of tubulescontaining an apoptotic nucleus in sham-treated controls (sham),ischemic-reperfused (I/R), and ischemic-reperfused mice treated withNGAL (+Ngal).

FIG. 14E shows an immunoblot of heme oxygenase-1 (HO-1) expression insham-treated (Sham), ischemic-reperfused (ATN), or ischemic-reperfusedNGAL-treated kidneys (ATN+Ngal). Recombinant HO-1 (HO-1) and rat cortexare included for comparison. GAPDH is the loading control.

FIG. 15 shows an immunoblot of clearance of NGAL protein in blood andurine of mice following intraperitoneal injection of 100 μg NGAL.

FIG. 16A shows fluorescent-labeled (Alexa568) NGAL localized to largevesicles in the proximal tubule (bottom panel) but not in the glomerulusor medulla (small top panels). Uncoupled fluorescent dye did not labelthe kidney.

FIG. 16B shows Alexa568-NGAL co-localized with FITC-dextran in S1 and S2segments of the proximal tubule.

FIG. 16C shows SDS-PAGE separation of ¹²⁵I-NGAL, with full length and a14 kDa fragment of NGAL found in the kidney 1 and 5 hours afterinjection.

FIG. 16D shows radioautograph of kidney one hour after intraperitonealinjection of ⁵⁵Fe loaded siderophore-NGAL. Radioactive decay is found inthe cortex and is associated with the apical zones of proximal tubulecells

FIG. 16E show radioautograph of kidney one hour after intraperitonealinjection of ⁵⁵Fe loaded siderophore-NGAL, with no radioactivity wasfound in the medulla.

FIG. 17A shows plasma creatinine in mice subjected to 30 minutes ofischemia. The first panel shows that holo-NGAL (>1 μg) from XL-1bacteria (containing siderophore) rescues renal function when introduced15 minutes prior to ischemia or within one hour after ischemia. However,NGAL is ineffective when administered later. The second panel shows thatapo-NGAL from BL-21 bacteria (siderophore free) is minimally active, butwhen loaded with enterochelin, the protein is protective. Both iron-free(apo-NGAL:Sid) and iron-loaded siderophores (apo-NGAL:Sid:Fe) haveprotective effect. In comparison, the gallium-loaded complex(apo-NGAL:Sid:Ga) was ineffective as was a single dose of DFO or thefree siderophore (Sid). Retinol Binding Protein (RBP), a lipocalin thatis also filtered and reabsorbed by the proximal tubule was ineffective

FIG. 17B shows immunoprecipitate preparations of NGAL. NGAL:Sid containsenterochelin, but not iron. NGAL:Sid:Fe contains siderophore and iron.

FIG. 18A shows that an iron-binding cofactor is present in urine. Bufferis mixed with ⁵⁵Fe (no protein), with apo-NGAL, apo-NGAL+siderophore, orapo-NGAL+siderophore+unlabeled iron. After a series of washes, ⁵⁵Fe isretained by apo-NGAL+siderophore but not by apo-NGAL or apo-NGAL ligatedby the iron-saturated siderophore, demonstrating that an unsaturatedsiderophore is required for retention of ⁵⁵Fe by NGAL.

FIG. 18B shows that when urine (<3,000 Da) is mixed with ⁵⁵Fe or withapo-NGAL, as indicated, and then washed three times on a 10 KDa filter,apo-NGAL+urine retains ⁵⁵Fe. ⁵⁵Fe retention was blocked by the additionof excess iron-citrate (Fe). Activity was also blocked by iron saturatedenterochelin (Sid:Fe).

FIG. 19 shows kidney biopsies obtained within 1 hour of transplantationfrom living related (panels 0 and 1) or cadaveric (panels 2 and 3)kidney transplants. Sections were stained with NGAL antibody. NGALexpression was significantly increased in the cadaveric group, whichunderwent a longer ischemic period.

FIG. 20 shows Western blots of urine samples obtained within 2 hours oftransplantation from living related (LRD, n=4) or cadaveric (CAD, n=4)kidney transplants, probed with NGAL antibody. NGAL expression in theurine was absent before the operation. NGAL expression was significantlyincreased in the CAD group compared to the LRD group

FIG. 21 shows quantitation of urinary NGAL by Western blots in LRDversus CAD, showing a significantly increased expression in CAD.

FIG. 22 shows the correlation of urinary NGAL obtained 2 hours after CADtransplantation with cold ischemia time. The degree of urinary NGALexpression correlates with ischemia time.

FIG. 23 shows the correlation of urinary NGAL obtained 2 hours after CADtransplantation with peak serum creatinine measured 2-4 days after theoperation. The degree of urinary NGAL expression correlates with peakserum creatinine.

FIG. 24 shows standard curves for NGAL ELISA with the linearrelationships obtained from 10 independent standard curves.

FIG. 25 shows serial serum NGAL measurements in patients who developedARF following cardiopulmonary bypass during surgery (CPB) (n=10).

FIG. 26 shows means±SD for serial serum NGAL levels in CBP patients whodeveloped ARF (squares) (n=10) versus those who had an uneventfulpostoperative course (diamonds) (n=30).

FIG. 27 shows serial urine NGAL measurements in CBP patients whodeveloped ARF following CPB (n=11).

FIG. 28 shows means±SD for serial urine NGAL levels in CBP patients whodeveloped ARF (diamonds) (n=11) versus those who had an uneventfulpostoperative course (squares) (n=30).

FIG. 29 shows the correlation between urine NGAL levels 2 hours afterCPB versus CPB time.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The accompanying sequence listings, which are incorporated in andconstitute a part of this specification, serve to explain the principlesof the invention.

SEQ:ID 01 is an example of a Primer sequence (forward primer, positions93-112) for mouse NGAL mRNA (Genbank NM_(—)008491).

SEQ:ID 02 is an example of a Primer sequence (reverse, positions576-557) for mouse NGAL mRNA (Genbank NM_(—)008491).

SEQ:ID 03 is an example of Sequences (forward, positions 415-434) formouse β-actin mRNA (Genbank X03672).

SEQ:ID 04 is an example of Sequences (reverse, positions 696-677) formouse β-actin mRNA (Genbank X03672).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “organ” means a differentiated biologicalstructure comprised of cells and tissues that perform a certain functionor functions in an organism, such as a mammal, including humans.Representative organs include, but are not limited to, the kidney,liver, heart, bone, cartilage, skin, lung, blood vessels, bladder,certix, stomach, intestine, pancreas, small intestine, colon, pancreasand brain, and portions or sections thereof.

As used herein, the term “renal injury” or “renal disease” shall includeacute (including but not limited to ischemia, ischemia-reperfusion,nephrotoxic, shock, stroke, sepsis, trauma, infection, inflammation) orchronic (including but not limited to hypertension, diabetes, heartfailure, lupus, infections, inflammations) kidney injuries orconditions.

The phrases “pharmaceutically acceptable,” “pharmacologicallyacceptable,” and “physiologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human.

The phrase “pharmaceutically acceptable carrier” as used herein means amaterial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting the compounds of the invention from one organ,or portion of the body, to another organ, or portion of the body withoutaffecting its biological effect. Each carrier should be “acceptable” inthe sense of being compatible with other ingredients of the compositionand not injurious to the subject. Some examples of materials which canserve as pharmaceutically-acceptable carriers include, but are notlimited to: any and all solvents, dispersion media, coatings,surfactants, antioxidants, preservatives (e.g., antibacterial agents,antifungal agents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art. Except insofar as any conventionalcarrier is incompatible with the NGAL, sideophore, or complex thereof,or other optional active agent or ingredient, its use in the therapeuticor pharmaceutical compositions is contemplated.

The phrase “therapeutically effective amounts” refers to those amountsof NGAL, siderophore, and mixtures thereof, or of other optional activeagents or ingredients, that is effective to produce beneficial results,particularly with respect to the treatments described herein, in therecipient, such as an animal or patient. Such amounts can be initiallydetermined by reviewing the published literature, by conducting in vitrotests or by conducting metabolic studies in healthy experimentalanimals. Before use in a clinical setting, it can be beneficial toconduct confirmatory studies in an animal model, typically a widelyaccepted animal model of the particular disease to be treated. Typicalanimal models for use in certain embodiments are rodent and murinemodels, which are economical to use and, particularly, because theresults gained are widely accepted as predictive of clinical value.

The term “derivative(s)”, in reference to NGAL, refers to chemicallymodified NGAL compounds, substances, inhibitors, or stimulators thatstill retain the desired effects on property(ies) of ischemia, renaltubule necrosis, nephrotoxicity, ischemic-reperfusion injury, and thelike. Such derivatives can include the addition, removal, orsubstitution of one or more chemical moieties on the parent molecule.Such moieties can include, but are not limited to, an element such ashydrogen, a halide, or a molecular group such as a methyl group. Such aderivative can be prepared by any method known to those of skill in theart. The properties of such derivatives can be assayed for their desiredproperties by any means described or known to those of skill in the art.

The term “analog” includes a structural equivalent or mimetic, asunderstood by those of skill in the art.

A “patient”, “recipient”, or “subject” means an animal or organism, suchas a warm-blooded animal or organism. Illustrative animals include,without limitation, mammals, for example, humans, non-human primates,pigs, cats, dogs, rodents, horses, cattle, sheep, goats and cows. Theinvention is particularly suitable for human patients and subjects.

An “inhibitor” means a compound, substance or agent that produces anymeasurable decrease in the activity, function, production, or secretionof a protein or biological compound, or in the translation of mRNA, inor from a cell.

As used herein in connection with transplanted and grafted organs, a“reduction” of ischemic injury or ischemic-reperfusion injury refers toany measurable decrease, diminution or reversal of damage to organs that(i) are stored, e.g., in preservation solution or in a cadaver, or (ii)are transplanted or grafted into a patient. Similarly, “reducing” refersto any measurable decrease or diminution, or a complete inhibition ofdamage, injury, or insult to organs that are stored, transplanted, orgrafted into a patient.

The words “a” and “an” as used herein refers to “one or more”. Morespecifically, the use of “comprising,” “having,” or other open languagein claims that claim a combination or method employing an object,denotes that “one or more of the object” can be employed in the claimedmethod or combination.

The present invention provides neutrophil gelatinase-associatedlipocalin, or NGAL, for use in methods of treating, reducing, orameliorating ischemic injury, ischemic-reperfusion injury, and atoxin-induced injury, to an organ such as the kidney. The presentinvention also provides the use of NGAL in methods of treating,reducing, or ameliorating acute kidney injuries (including but notlimited to shock, trauma, stroke, sepsis, infection, inflammation,stones, and surgeries) and chronic kidney injuries (including but notlimited to hypertension, diabetes, heart failure, lupus, inflammation,glomerulonephritis and interstitial nephritis). In accordance with theinvention, yet without wishing to be bound by theory, NGALadministration has been found to affect tubule cell death so as to limitapoptotic tubule cell death, i.e., apoptosis, and to enhancere-epithelialization, i.e., the recovery of viable cells followingischemia in the kidney involving de-differentiation and proliferation ofviable cells and re-establishment of the epithelial phenotype followingischemia-reperfusion injury. NGAL administration has also been shown toreduce increases in serum and plasma creatinine levels after ischemicinjury.

Human NGAL, a 25 kDa protein that is covalently bound to gelatinase fromhuman neutrophils, is expressed at very low levels in several humantissues, including kidney, trachea, lungs, stomach, and colon. NGALexpression is markedly induced in and secreted by stimulated epithelia.For example, NGAL concentrations are elevated in the serum of patientswith acute bacterial infections, the sputum of subjects with asthma orchronic obstructive pulmonary disease, and the bronchial fluid from theemphysematous lung. NGAL is also one of the maximally-induced genes inthe kidney following early ischemic injury. These data are derived fromanalyses of mRNA by gene chip, implicating that the damaged kidneysynthesizes NGAL. Other studies have shown that NGAL can be found atelevated levels in the serum of human patients with inflammatorydiseases. Hence we have evaluated the incidence of expression of NGAL inhuman ATN compared to chronic forms of renal disease. NGAL is highlyexpressed in clinically defined ATN and appears in the proximal tubulein biopsied human kidney. Although less abundant, NGAL is also expressedin the kidney in several forms of chronic kidney disease. A mouse modelof ischemia/reperfusion induced ATN also expresses NGAL at very highlevels. Microgram quantities of injected NGAL provided dramaticprotection against ATN as measured by plasma creatinine and by thehistology of the kidney. The protection of the kidney was due to thedelivery to the proximal tubule of NGAL protein containing the bacterialsiderophore.

The preparation of a pharmaceutical composition or formulationcomprising NGAL is known to those of skill in the art in light of thepresent invention, as exemplified by Remington's PharmaceuticalSciences, 18th Ed., Mack Printing Company, 1990. Moreover, for animal(e.g., human) administration, it will be understood that preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by the FDA Office of Biological Standards.

Previously, the role of NGAL was unclear; however, NGAL has now beenidentified as an iron-transporting protein during nephrogenesis. Despiteits high affinity for siderophores and for iron, NGAL can deliver ironto the cytoplasm. The likely mechanism for iron delivery from NGAL is byendocytosis. Fluorescent NGAL protein is endocytosed, and thistrafficking is blocked by 4° C. temperatures. Acidification of thesevesicles can be necessary for iron release from NGAL because agents thatinhibit acidification blocked iron uptake. Moreover, addition of NGAL tocells expressing a fluorescent iron reporter in the cell cytoplasmstimulated iron-activated changes in reporter expression, confirmingthat NGAL can serve as an iron donor. Notably, the pathway for NGALendocytosis differed from the pathway taken by holo-transferrin in cellsin culture. At steady state, holotransferrin trafficked to rab-5 orrab-7 recycling vesicles (rab5 and rab7 are markers for early and lateendosomes, respectively), while NGAL trafficked to late endosomes and ina small percentage to lysosmes. These data demonstrate that NGAL canserve in an iron delivery pathway when it contains a siderophore.

The actions of NGAL in vivo might differ from its pharmacologicaleffects because the critical siderophore in vitro is a bacterialproduct. A number of analyses have indicated the possibility ofendogenous low molecular weight co-factors for iron transport. Theseinclude citrate and related compounds, but also iron transportactivities in the molecular weight range of 1000 Da. To determinewhether such a co-factor might also be present in the kidney, we mixedapo-NGAL from BL21 bacteria with urine samples. While the urine itselffailed to trap ⁵⁵Fe, and apo-NGAL diluted in salt solutions failed totrap ⁵⁵Fe, dilution of NGAL in urine permitted retention of ⁵⁵Fe. Thisfinding suggests that a cofactor is present in urine that permitsNGAL-iron interactions.

In embodiments of the present invention, the exogenous administration ofNGAL can ameliorate the structural damage inflicted byischemia-reperfusion injury. Both apoptosis and necrosis can besignificantly blunted. Without wishing to be bound by theory, themechanism by which NGAL inhibits apoptosis in the ischemic conditionincludes an anti-apoptotic effect analogous to that of heme oxygenase 1(HO-1), which facilitates the extracellular transport of iron, therebylimiting iron-driven oxidant stress in the intracellular compartment (C.D. Ferris et al., 1999, Nat. Cell Biol., 3:152-157). As a carrier of anyof various siderophores, NGAL also facilitates the removal of excessintracellular iron, thereby limiting oxidant-mediated apoptosis of renaltubule cell death following ischemia-reperfusion injury. With respect tonecrosis, the response of the kidney following ischemia-reperfusioninjury can occur by a two-stage process, namely initiation of apoptosisfollowed by a necrotic cell death. In addition to limiting iron-mediatedoxidative-stress, apoptosis inhibition by NGAL can be effective inpreventing the secondary necrosis aspect of the process. By the processof apoptosis inhibition, NGAL can be effective in a variety of kidneydiseases that are well known to be associated with increased apoptosis,including but not limited to ischemia, ischemia-reperfusion,nephrotoxins, polycystic kidney disease, obstruction, inflammation, andglomerular diseases.

NGAL protein is composed of eight β strands which form a β-barrel or acalyx. The calyx binds and transports low molecular weight chemicals,including siderophores found in urine and/or produced by bacteria. Thebest evidence for NGAL's ligand-binding properties comes fromcrystallographic studies, which demonstrated a bacterial siderophore(enterochelin) in the β-barrel. NGAL binds the siderophore with highaffinity (0.4 nM) and the siderophore traps iron with high affinity(10⁻⁴⁹M). The stoichiometry of protein:siderophore:iron is 1:1:1, asdemonstrated by binding studies and x-ray crystallography. When thesiderophore was loaded with iron, the NGAL complex donated iron toembryonic mesenchyme in vitro and to cell lines, and when thesiderophore was iron-free, the NGAL complex chelated iron. NGAL wasendocytosed by many cell types, and trafficked to a late endosomalcompartment that differed from the transferrin compartment. Donation ofiron took place in an endosomal compartment. Because NGAL is the firstmammalian protein found to bind bacterial siderophores, it can also beencalled siderocalin.

Siderophores are small protein molecules that scavenge iron from theenvironment, having a low molecular weight ranging from about 500 toabout 1000 MW. Siderophores can chelate ferric iron. Iron-catalyzeddamage is thought to be one of the earliest events in kidney dysfunctionfollowing an ischemic, ischemic-reperfusion, or toxin-induced injury,and is likely to be important in the early stages of damage to otherorgans, including the heart and the liver. Chelating iron withsiderophores can blunt the damage to these organs.

Siderophores can be synthetic or naturally-occurring products harvestedfrom bacterial cultures, and are commercially available. Siderophoresare avidly taken up by NGAL when mixed together under physiologicalconditions in a wide variety of commonly used buffers including 10 mMTris or Phosphate-buffered Saline. Typically, siderophores can be addedin excess to a known quantity of NGAL protein. NGAL molecules will bindto siderophore molecules such that each complex will contain onemolecule of each species. The 1:1 complexes of NGAL:siderophore arewashed to remove the excess unbound siderophore molecules, and can thenbe further processed for use in the practice of the invention.Alternatively, equimolar amounts of siderophore and NGAL molecules canbe combined and incubated to allow binding. Exogenous siderophorescontemplated for use in the invention include, but are not limited toenterochelin, carboxymycobactin, aminochelin, desferrioxamine,aerobactin, arthrobactin, schizokinen, foroxymithine, pseudobactins,ncocnactin, photobactin, ferrichrome, hemin, achromobactin,achromobactin, rhizobactin, and other bacterial products, as well ascitrate and synthetic analogs and moieties and others that can beproduced using organic chemistry processes. Endogenous siderophores canalso be complexed to NGAL in vivo, as will be described in examples ofthe methods for use.

The methods of the present invention provide certain advantages for thepatient. Acute renal failure secondary to ischemic injury remains acommon problem, with limited and unsatisfactory therapeutic options. Theidentification of factors that inhibit, reduce, or oppose tubule celldeath (necrosis/apoptosis) and/or enhance the recovery phase (involvingde-differentiation and proliferation of viable renal tubule cells andre-establishment of the epithelial phenotype) can serve as noveltherapeutic options. In accordance with this invention, NGAL, both aloneand together with siderophores, advantageously exhibits theabove-mentioned desirable and cytoprotective properties. Exogenouslyadministered NGAL has been demonstrated to limit the morphologic andfunctional consequences of ischemia-reperfusion injury in a mouse model,by a combination of limiting apoptotic tubule cell death and enhancingre-epithelialization.

In an embodiment of the invention for treating, reducing, orameliorating a toxin-induced injury, the toxin and/or the therapeuticthat is toxic, can include an antibiotic, an anti-inflammatory agent, anantifungal agents, a radio-contrast agent, a pharmaceutical, achemotherapeutic agent, a test drug, a medicament substance, ornaturally-occurring, commercial and industrial chemicals and minerals.Specific toxins and nephrotoxins include, but not limited to, a cancerchemotherapeutic such as cisplatin, mitomycin, cyclophosphamide,isosfamide, and methotrexate, an antibiotic including gentamicin,vancomycin, and tobramycin, an antifungal agent, such as amphotericin,an anti-inflammatory agent, such as an NSAID, an immunosuppressant, suchas cyclosporine and tacrolimus, other medicaments, commercial andindustrial chemicals, such as hydrocarbons, chlorocarbons andfluorocarbons, and minerals such as arsenic, mercury, bismuth and lead.Other nephrotoxic compounds can include an aminoglycoside, foscarnet,pentamidine, vancomycin, neomycin, nitrous oxide, isoflurane, kanamycin,and cyclophosphamide.

In accordance with embodiments of the invention, NGAL can beadministered prior to, during (at the same time as), or followingischemia, ischemic-reperfusion injury, organ transplant, ATN, toxinadmistration, and the like, as described herein. More particularly, NGALcan be administered to the patient from about 30 minutes to about 90minutes before an organ is transplanted. It is also contemplated thatthe compositions can be administered at times outside the range of 30 to90 minutes.

The invention also includes a method of administering from about 1 toabout 200 mg/kg body weight of NGAL to a patient, more typically fromabout 1 to about 100 mg/kg body weight. The amount of NGAL administeredto a patient can vary or fall out side of the ranges given above. Asdiscussed herein, the amount of NGAL administered to the patient canvary.

A composition of the present invention, such as a medicament orpharmaceutical composition, can typically comprise a level of NGALand/or sideophore of at least about 10 microgram/100 microliter ofcomposition, and more typically at least about 100 microgram/100microliter of composition.

A composition of the present invention can include different types ofpharmaceutically acceptable carriers, depending on whether they are tobe administered in solid, liquid or aerosol form, and whether they needto be sterile for such routes of administration as injection. Thepresent invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally,inhalation (e.g., aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the foregoing aswould be known to one of ordinary skill in the art.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of the conditionbeing treated, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient, and the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of the NGAL, siderophore andmixtures thereof, and of other optional active agents, in a compositionand appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions can comprise, forexample, at least about 0.1% of another optional active agent oringredient. In other embodiments, the active agent or ingredient cancomprise between about 2% to about 75% of the weight of the unit, moretypically between about 25% to about 60%, and any range derivabletherein. In other non-limiting examples, a dose amount of the activeagent or ingredient can comprise from about 1 microgram/kg body weightabout 500 milligram/kg body weight, more typically from about 5 mg/kgbody weight to about 100 mg/kg body weight.

In some instances, the composition can comprise various antioxidants toretard oxidation of one or more ingredient. Additionally, the preventionof the action of microorganisms can be brought about by preservativessuch as various antibacterial and antifungal agents, including but notlimited to parabens (e.g., methylparabens, propylparabens),chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The compositions can be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium including, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes),and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size by dispersion in carriers such as, forexample liquid polyol or lipids, by the use of surfactants such as, forexample, hydroxypropylcellulose, or combinations thereof such methods.In many cases it is typical to include isotonic agents, such as, forexample, sugars, sodium chloride or combinations thereof.

In other embodiments of the present invention, one can use eye drops,nasal solutions or sprays, aerosols or inhalants. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in typical embodiments, the aqueous nasal solutions usually are isotonicor slightly buffered to maintain a pH of about 5.5 to about 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, can be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments, the compositions are prepared for administrationby such routes as oral ingestion. In these embodiments, the solidcomposition can comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions can be incorporated directly with the food of the diet.Typical carriers for oral administration comprise inert diluents,assimilable edible carriers, or combinations thereof. In other aspectsof the invention, an oral composition can be prepared as a syrup orelixir, and can comprise, for example, at least one optional activeagent or ingredient, a sweetening agent, a preservative, a flavoringagent, a dye, a preservative, or combinations thereof. In otherembodiments, an oral composition can comprise one or more binders,excipients, disintegration agents, lubricants, flavoring agents, orcombinations thereof.

In certain embodiments, a composition can comprise one or more of thefollowing: a binder, such as, for example, gum tragacanth, acacia,cornstarch, gelatin or combinations thereof, an excipient, such as, forexample, dicalcium phosphate, mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate orcombinations thereof, a disintegrating agent, such as, for example, cornstarch, potato starch, alginic acid or combinations thereof, alubricant, such as, for example, magnesium stearate, a sweetening agent,such as, for example, sucrose, lactose, saccharin or combinationsthereof, a flavoring agent, such as, for example peppermint, oil ofwintergreen, cherry flavoring, orange flavoring, etc., or combinationsof the foregoing. When the dosage unit form is a capsule, it cancontain, in addition to materials of the above type, carriers such as aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar or both.

Additional formulations that are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general forsuppositories, traditional carriers can include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories can be formed from mixtures containing, forexample, the active agent or ingredient in the range of about 0.5% toabout 10%, and typically about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active compounds into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the typicalmethods of preparation are vacuum-drying or freeze-drying techniquesthat yield a powder of the active compound plus any additional desiredingredient from a previously sterile-filtered liquid medium thereof. Theliquid medium should be suitably buffered if necessary and the liquiddiluent first rendered isotonic prior to injection with sufficientsaline or glucose. The preparation of highly concentrated compositionsfor direct injection is also contemplated, where the use of DMSO assolvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active compounds to a small area.

The composition should be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatexotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

Experimental Protocols

The invention will be better understood through examples illustratingits use and efficacy. The experimental protocols described below will bereferenced in the examples that follow.

1. Expression, Purification, and Radiolabeling of Recombinant Human andMouse NGAL:

Full length mouse NGAL cDNA was cloned into the pGEX expression vector,expressed as a fusion protein with glutathione S-transferase (GST) inbacteria, and purified using glutathione-sepharose columns (Amersham)followed by thrombin cleavage as previously by Bundgaard J et al.,Biochem Biophys Res Commun 202: 1468-1475, 1994; Yang J et al., Mol Cell10: 1045-1056, 2002; and Del Rio M et al., J Am Soc Nephrol 15: 41-51,2004. Purified NGAL was made endotoxin-free with using the Detoxi-Gelendotoxin removing column (Pierce) as recommended by the manufacturer.Proteins were analyzed by SDS-PAGE followed by Coomassie blue stainingor by Western blotting with a polyclonal antibody to NGAL as describedby Mishra et al., J Am Soc Nephrol 14: 2534-2543, 2003. Proteinconcentrations were determined using the Bradford assay. A single cleanpolypeptide of the predicted size was detected, as shown in FIG. 1.

2. Expression and Purification of Recombinant Human and Mouse NGAL

Recombinant human and mouse GST-NGAL were expressed in BL21 or XL1-Bluestrains of E. coli (Stratagene) with additional ferric sulfate (50 MicroMolar, Sigma-Aldrich Co.). NGAL was isolated using Glutathione Sepharose4B beads (Amersham Biosciences), eluted by thrombin cleavage(Sigma-Aldrich Co.; St. Louis, Mo.) and then further purified by gelfiltration (Superdex75, SMART system, Amersham Biosciences) and examinedby Coomassie gels (Biorad). BL-21 derived NGAL was loaded with iron freeor iron saturated enterochelin, a siderophore (EMC Microcollections)using a 5 fold molar excess. Unbound siderophore (0.7 KD) was removed bywashing (Microcon YM-10) with PBS. To produce ⁵⁵Fe or gallium (Ga)loaded NGAL we incubated the iron-free enterochelin NGAL complex withequimolar ⁵⁵Fe (18mCi) or Ga in NaCl (150 mM) Hepes (20 mM; pH 7.4) andthe complex was washed 3 times (10K filter). Iodobeads (Pierce) wereused to label NGAL with ¹²⁵I and unincorporated ¹²⁵I was removed by gelfiltration (PD-10 column) followed by extensive dialysis (7 kDa cut offmembrane, Pierce) against PBS. Alexa-568 and fluorescein isothiocyanate(Molecular Probes) was coupled to NGAL, according to the manufacturer,and then extensively dialyzed. Protein concentration was determined byCoomassie gels in comparison with bovine serum albumin standard.

3. NGAL Injections:

Purified endotoxin-free NGAL was administered either intravenously intomice via tail vein injections, subcutaneously, or intraperitoneally. Inpreliminary studies, animals were treated with three differentconcentrations of NGAL (50, 100, or 250 μg of a 250 μg/100 solution),subjected to 30 minutes of bilateral renal artery clamping one hourlater, and examined after 24 hours of reflow. When compared to animalspre-treated with an equal volume (100 μl) of saline, only the groupgiven 250 μg of NGAL exhibited a significant protection from the tubulardamage and azotemia. All subsequent studies as reported here werecarried out using the 250 μg dose of NGAL. Comparisons were made betweenfive different animal groups: non-ischemic controls (n=8), ischemiccontrols pre-treated with saline alone (n=8), NGAL pre-treated one hourprior to renal artery clamping (n=6), NGAL treated during renal arteryclamping (n=6), and NGAL treated one hour post renal artery clamping(n=6).

4. Human Studies:

Healthy volunteers and patients diagnosed with either acute or chronicrenal failure were analyzed for NGAL protein levels in urine and serum.Acute renal failure (ARF) was diagnosed by a doubling of the serumcreatinine in less than 5 days. The presumed etiology of ARF includedsepsis which was defined by the presence of at least two of thefollowing criteria: positive blood cultures or evidence of localinfection in the lung, skin or urinary tract and fever or an elevatedWBC count. Some of these patients required blood pressure support. Otheretiologies of ARF included hypotension due to bleeding or heart failure,nephrotoxins, or post-transplant ischemia. The definition of chronicrenal failure (CRF) was a serum creatinine greater than 2 mg/dl, butunchanged during at least the prior 2 months. The presumed etiologies ofCRF included obstructive uropathy, chronic interstitial nephritis, anddiabetes. Samples of blood and urine were collected from patientsevaluated at Columbia University Medical Center and at Kyoto UniversityHospital with approval of both Institutional Review Boards and thenanalyzed in a blinded fashion.

5. Measurement of NGAL:

An anti-mouse NGAL polyclonal antibody was raised in rabbit and thenpurified on a column of Sepharose 4 fast flow beads (AmershamBiosciences) coupled to recombinant mouse NGAL (see below) followed byelution at pH 2.5. Monoclonal anti-human NGAL (AntibodyShop) was alsoused to detect NGAL. Human NGAL was better recognized by the monoclonalantibody while mouse NGAL was recognized only by the affinity-purifiedpolyclonal.

Human blood samples were initially collected in citrate, EDTA orheparin, but since all of these preparations showed similar NGALimmunoreactivity, human serum and mouse plasma are collected in theexamples described below. The samples were centrifuged through a 100 KDacut-off filter (YM-100, Amicon) and the flow-through used forimmunoblot. In patients undergoing hemodialysis, samples were takenimmediately before dialysis. Fresh urine samples were centrifuged at lowspeed and then used without further concentration.

6. Pathologic Specimens:

Pathological specimens included ischemic ATN (10 cases), toxic ATN (11cases-(5) antibiotics, (2) zoledronate, (1) carboplatinum, (2)non-steroidal anti-inflammatory agents, and (1) hemoglobinuria), andglomerulopathies (10 cases—including diabetic, anti-GBM, pauci-immunecresentic glomerulonephritis, IgA nephropathy, minimal change, focalsegmental glomeruloscerosis), and also normal kidneys (3 cases).Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm) andsubjected to antigen retrieval using microwave in a citrate buffer(pH6.0) for 30 min. Endogenous peroxidase was blocked with 5% H₂O₂ for30 min, followed by blocking in 10% goat serum/1% BSA. Affinity purifiedanti-mouse NGAL (0.4 μg/ml) was applied overnight at 4C, followed bybiotinylated goat anti-rabbit IgG (1:100, Vector) and avidin-HRP, eachfor 30 minutes. Slides were developed with DAB/0.3% H₂O₂ for 2.5 minutesand counterstained with hematoxylin. Non-immune rabbit IgG (0.4 μg/ml;Vector) was used as a control.

7. NGAL Trafficking

To detect delivery of NGAL to the kidney rNGAL (10 or 100 μg), Alexa568-NGAL (100 μg), ¹²⁵I-NGAL (10 mg, 2×10⁶ cpm), or ⁵⁵Fe loadedenterochelin-NGAL (10 μg, 1×10⁶ cpm) was injected into the peritoneumand blood, urine, kidney and liver samples were obtained. NGAL wasdetected by immunoblot. Alexa-568 NGAL was detected by confocalmicroscopy (LSM Meta Detector) and NGAL mediated iron trafficking wasdetected by scintillation counter and by light microscopicradioautography of Epon embedded kidneys. Slides were exposed toemulsion (Polyscience) for 1 week and then developed with Microdol andcounterstained with Toluidine blue. To detect lysosomes in the proximaltubule, mice were injected with Fluorescein Dextran (46 kD; 0.5 mg;Sigma) 24 hours before Alexa 568 NGAL injections. LAMP1 (Santa Cruz) wasdetected in cryostat sections of 4% paraformaldehyde fixed kidneys.

8. Mouse Model of ATN or Ischemia/Reperfusion Injury:

Male C57BL/6 mice (20-25 gr; Charles River) were anesthetized withintraperitoneal pentobarbital (50 mg/kg) and placed on a heating padunder a warming light to maintain 37© core body temperature. Kidneyswere exposed through an abdominal section and the right kidney waseither removed or its vascular pedicle and ureter ligated. The vascularpedicle of the left kidney or both kidneys was clamped by amicroaneurysm clip (Kent Scientific) for 30 minutes after rightnephrectomy. This period of ischemia generated reproducible renal injurybut minimized mortality. During the procedure, PBS (0.5 ml) was used todampen the peritoneum. The animal was closed with 5-0 Nylon. Saline,NGAL, retinol-loaded retinol binding protein (RBP), enterochelin, ordesferroxamine mesylate (DFO) were injected into the peritoneum orsubcutaneously 15 min. prior to ischemia or 1-2 hr after reperfusion.

After 6 or 24 hr of reperfusion, heparinized plasma, urine and kidneysamples were obtained to measure NGAL (polyclonal 1:500), Hemeoxygenase-1 (Stressgen, 1:2000), E-cadherin (BD Transduction Labs,1:2000), N-cadherin (BD Transduction Labs, 1:3000) and GAPDH (ChemiconInternational, 1:3000) using immunoblots. Plasma was also used forcreatinine and blood urea nitrogen colorimetric assays. Sagittalsections of the kidney were fixed in 4% formalin, or were snap frozenfor mRNA and protein analysis. Paraffin-embedded sections (5 μm) werestained with hematoxylin-eosin or by an in situ kit (Fluorescein-TUNEL,Roche) for apoptotic nuclei or for total nuclei (Toprol, MolecularProbes). For cell proliferation analysis, BrdU was injected into theperitoneum 1 hour before sacrifice, and cryostat sections were stainedwith anti-BrdU (Roche) according to the manufacturer.

For some studies, the mice were allowed to recover in a warmed cage, andtimed urine collections were obtained. After various reperfusionperiods, the animals were then re-anesthetized, the abdominal cavityopened, and blood obtained via puncture of the inferior vena cava formeasurement of serum creatinine by quantitative colorimetric assay. Themice were killed, the kidneys perfusion fixed in situ with 4%paraformaldehyde in PBS, and both kidneys harvested. One half of eachkidney was snap frozen in liquid nitrogen and stored at −70° C. untilfurther processing; a sample was fixed in formalin, paraffin-embedded,and sectioned (4 μm). Paraffin sections were stained withhematoxylin-eosin and examined histologically. The other half of eachkidney was embedded in OCT compound (Tissue-Tek) and frozen sections (4μm) obtained for immunohistochemistry.

9. Real-Time PCR:

Total RNA was extracted from mouse kidneys using RNeasy mini kit(Qiagen) with on-column DNase digestion according to the manufacturer'sinstructions. The cDNA template was synthesized using Omniscript ReverseTranscriptase and oligo-dT primer (Qiagen). The PCR reaction was carriedout using iQ SYBR green super mix and MyiQ single-color real-time PCRdetection system (Biorad) with incubation times of 2 min at 95° C.,followed by 40 cycles of 95° C./30 s and 60° C./30 s. Specificity of theamplification was checked by melting curve analysis and by agarose gelelectrophoresis. Primer sequences for mouse NGAL mRNA (GenbankNM_(—)008491) were CTCAGAACTTGATCCCTGCC (forward primer, positions93-112) and TCCTTGAGGCCCAGAGACTT (reverse, 576-557). Sequences for mouseβ-actin mRNA (Genbank X03672) were CTAAGGCCAACCGTGAAAAG (forward,415-434) and TCTCAGCTGTGGTGGTGAAG (reverse, 696-677). Each plateincluded a dilution series of standard sample, which was used todetermine mRNA quantities. The NGAL mRNA content was normalized byβ-actin mRNA.

10. Iron Binding Co-Factor

Cofactor-dependent iron binding to NGAL was measured in 150 mM NaCl-20mM Hepes (pH7.4) buffer (100 μl) with apo-NGAL (10 μM), ⁵⁵Fe (1 μM), anda low molecular weight fraction (<3 Kd) of mouse urine (0-30 μl) andincubated 70 min. at room temperature. The urine fraction was obtainedby passing fresh urine sequentially through 10 kDa and 3 kDa membranes(YM-10 and YM-3, Amicon). The mixture was then washed three times on 10kDa membrane (YM-10, Amicon). Iron-free enterochelin-loaded NGAL (ratherthan NGAL without siderophore) served as a positive control for ironcapture. Ferric citrate (1 mM) or iron-loaded enterochelin (Sid:Fe, 50μM) were used as competitors of ⁵⁵Fe binding.

11. NGAL Immunohistochemistry:

For NGAL detection, frozen kidney sections were permeabilized with 0.2%Triton X-100 in PBS for 10 min, blocked with goat serum for 1 hr, andincubated with primary antibody to NGAL (1:500 dilution) for 1 hr.Slides were then exposed for 30 min in the dark to secondary antibodiesconjugated with Cy5 (Amersham, Arlington Heights, Ill.), and visualizedwith a fluorescent microscope (Zeiss Axiophot) equipped with rhodaminefilters.

12. Histopathology Scoring:

Kidney sections of 4 microns were stained with hematoxylin-eosin andscored for histopathologic damage to the tubules in a blinded fashion,as previously described by Yokota N. et al., Am J Physiol Renal Physiol285: F319-F325, 2003 and Kjeldsen L. et al., Biochim Biophys Acta 1482:272-283, 2000. Each parameter was assessed in five high power fields(40×) in the inner cortex and outer medullary regions (where the tubulardamage was most evident), and an average determined for each section.The parameters included tubule dilatation, tubule cast formation, andtubule cell necrosis. Each parameter was scored on a scale of 0 to 4,ranging from none (O), mild (1), moderate (2), severe (3), to verysevere/extensive (4).

13 Apoptosis Assays:

For the TUNEL assay to detect apoptotic nuclei, we utilized the ApoAlertDNA Fragmentation Assay Kit (Clontech). Paraffin sections weredeparaffinized through zylene and descending grades of ethanol, fixedwith 4% formaldehyde/PBS for 30 min at 4° C., permeabilized withproteinase K at room temperature for 15 min and 0.2% triton X-100/PBSfor 15 min at 4° C., and incubated with a mixture of nucleotides and TdTenzyme for 60 min at 37° C. The reaction was terminated with 2×SSC, thesections washed with PBS, and mounted with Crystal/mount (Biomeda,Foster City, Calif.). TUNEL-positive apoptotic nuclei were detected byvisualization with a fluorescent microscope. Only cells that displayedthe characteristic morphology of apoptosis, including nuclearfragmentation, nuclear condensation, and intensely fluorescent nuclei byTUNEL assay, were counted as apoptotic. Merely TUNEL positive cells, inthe absence of morphologic criteria, were not considered apoptotic.Slides were examined in a blinded fashion, and apoptosis was quantifiedby counting the number of TUNEL positive nuclei per 100 cells counted inan average of five high power (40×) fields in each section.

14. Proliferation Assays:

For detection of proliferating cells, sections were incubated with amonoclonal antibody to Proliferating Cell Nuclear Antigen (PCNA, 1:500dilution, Upstate Biotechnology), and detection accomplished byimmunoperoxidase staining as recommended by the manufacturer (ImmunoCruzStaining System, Santa Cruz Biotechnology). Slides were examined in ablinded fashion, and proliferation was quantified by counting the numberof PCNA positive cells per 100 cells counted in an average of five highpower (40×) fields in each section.

15. Statistical Analysis:

The SPSS software (version 8/0) was employed to generate univariatestatistics for each continuous variable, including means, standarddeviations, distributions, range, and skewness. The data were examinedfor normality and equality of distribution. One way ANOVA was employedto compare means±SD of continuous variables among different treatmentgroups. The Kruskal-Wallis ANOVA on Ranks was used for non-normallydistributed data. To identify the group or groups that differed from theothers, a multiple comparison procedure was used (Tukey test or Dunn'sMethod depending on the normality of distribution). A p value<0.05 wasconsidered statistically significant. NGAL levels in humans were logtransformed for statistical analysis. The data were analyzed by one-wayANOVA with Bonferroni's post-test to compare mean values across groups.The Jablonski score of kidney damage was analyzed by the Kruskal-Wallistest with Dunn's post-test.

EXAMPLES

The following examples are provided to more fully describe the practiceof the invention in its various embodiments. Experimental protocolsprovided above are used as indicated in the examples.

Example 1

Intravenous NGAL is rapidly taken up by proximal tubule cells in vivo.Purified NGAL was delivered to its putative site of action, namely theproximal tubule. Mice received intravenous NGAL (250 μg in 100 μlsaline) or an equal volume of saline alone, subjected toischemia-reperfusion injury, and the kidneys and urine examined atvarious time periods, as shown in FIG. 2. Non-ischemic saline controlanimals had no NGAL (upper left panel), while non-ischemic NGAL-treatedanimals had NGAL (lower left panel). Saline-injected animals were devoidof kidney NGAL at one hour (upper center panel). Endogenous NGAL wasdetected in saline-treated animals at 3 hours after ischemic-reperfusioninjury (upper right panel). In contrast, within one hour of NGALinjection, it was easily detected in a punctate cytoplasmic distributionpredominantly in the proximal tubules (lower center panel), and wasstill seen at 3 hours (lower right panel). Identification of proximaltubules in these sections was based on location and morphology. Thisrepresents uptake of injected NGAL following ischemic injury, since NGALwas not detected at the one hour reflow period in saline-injectedanimals. In addition, NGAL was detected in the urine within one hour ofinjection, as shown in FIG. 3.

Example 2

Intravenous NGAL rapidly appears in the urine following administrationand ischemic-reperfusion injury. Urine from the animals of Example 1 wasexamined at various time periods. Saline-injected animals were devoid ofkidney or urinary NGAL at the 1 hour reflow period, and NGAL was justdetectable at the 3-hour reflow period, as shown in FIG. 3 (left panel).The 3 hour data represents the endogenous response of kidney tubulecells to ischemic injury. In contrast, in animals injected with NGAL andsimultaneously subjected to ischemia-reperfusion injury, NGAL was easilydetected in the kidney and urine with 1 hour of reflow, as shown inFIGS. 3 (right panel).

Example 3

NGAL ameliorates the histopathologic damage to tubules induced byischemia-reperfusion injury. NGAL administered one hour before, during,or even one hour after ischemia resulted in a significant decrease inthe histopathologic damage to tubules. Representative kidney sectionsobtained at 24 hours of reflow and stained with hematoxylin-eosin areshown in FIG. 4. While the non-ischemic controls (Non-Ischemic panel)displayed normal histology, animals pre-treated with saline alone(Saline Pre-treated panel) (100 μl, volume of diluent) displayedextensive features of acute tubular necrosis, including tubulardilatation, tubular cast formation, and necrotic cells. In contrast,NGAL-treated kidneys displayed an attenuated histopathologic response.This was most evident in animals pre-treated with NGAL (NGAL Pre-treatedpanel), but was also evident when the NGAL was administered during (NGALDuring Isch panel) or even one hour after (NGAL After Isch panel) theischemic injury. In order to quantify this response, kidney sectionswere scored for histopathologic damage to the tubules in a blindedfashion. The results are illustrated in FIG. 5A-5C. In all threeparameters examined, dilatation (FIG. 5A), casts (FIG. 5B), and cellnecrosis (FIG. 5C), all three modalities of NGAL treatment (before,during, or after ischemia) resulted in a significantly improved scorewhen compared to controls. This difference was most striking in animalspre-treated with NGAL, followed in a graded fashion by findings inanimals treated with NGAL during ischemia or after the ischemic insult.However, the structural protection was not complete, and even animalspre-treated with NGAL did display some degree of histopathologic damage,which was completely absent from non-ischemic controls.

Example 4

NGAL ameliorates the reduction in kidney function induced byischemia-reperfusion injury. NGAL administered one hour before, during,or even one hour after ischemia resulted in a significant decrease inthe serum creatinine measured at 24 hours of reflow, as shown in FIG. 6.While the non-ischemic controls (Non Isch) displayed serum creatinine(0.65±0.13 mg/dl), animals pre-treated with saline alone (Pre Sal) (100μl, volume of diluent) displayed a significant increase in serumcreatinine (2.6±0.28 mg/dl). In contrast, NGAL-treated kidneys displayedan attenuated functional response. This was most evident in animalspre-treated with NGAL (1.25±0.3 mg/dl), but was also evident when theNGAL was administered during (Dur NGAL) (1.5±0.2 mg/di) or even one hourafter (Post NGAL) (1.95±0.2 mg/di) the ischemic injury. However, thefunctional protection was not complete, and even animals pre-treatedwith NGAL did display a significant increase in serum creatinine whencompared to non-ischemic controls.

Example 5

NGAL ameliorates the apoptotic, tubule cell death induced byischemia-reperfusion injury. The structural and functional protectionobserved with exogenous NGAL administration was a result of decreasedapoptosis. Representative kidney sections obtained at 24 hours of reflowand subjected to TUNEL assay are shown in FIG. 7 at low (left column)and high (center column) magnifications. While the non-ischemic controlsdisplayed a minimal incidence of apoptosis (2.2±0.5 cells per hundred(%) cells examined), animals pre-treated with saline alone (100 μl,volume of diluent) displayed a significantly greater number of apoptotictubule epithelial cells (12.6%±2.2), as shown quantitatively in FIG. 8(left panel). In contrast, NGAL-treated kidneys displayed an attenuatedapoptotic response. This was most evident in animals pre-treated withNGAL (6.7%±1.6), but was also evident when the NGAL was administeredduring (7.6%±0.8) or even one hour after (8.5%±0.8) the ischemic injury.However, the protection from apoptotic cell death was not complete, andeven animals pre-treated with NGAL did display a significantly greaterdegree of apoptotic damage when compared to non-ischemic controls.

Example 6

NGAL enhances tubule cell proliferation following ischemic injury.Representative kidney sections obtained at 24 hours of reflow andstained with an antibody to PCNA are shown in FIG. 7 (right column).While the non-ischemic controls displayed a minimal incidence ofproliferating cells (1.9%±0.4 cells per hundred cells examined), animalspre-treated with saline alone (100 μl, volume of diluent) displayed asmall but significant increase in the number of PCNA-positive tubuleepithelial cells (4.4%±1.2), as shown quantitatively in FIG. 8 (rightpanel). In contrast, NGAL-treated kidneys displayed a marked increase inproliferating cells. This was most evident in animals pre-treated withNGAL (19.1%±2.1), but was also evident when the NGAL was administeredduring (14.9%±1.2) or even one hour after (14.5%±1.2) the ischemicinjury.

Example 7

NGAL tilts the balance of tubule cell fate towards survival followingischemic injury. The overall tubule cell fate following ischemic injurywas estimated using a one-way ANOVA to compare means±SD of proliferationand apoptosis among the different treatment groups at 24 hours ofreflow. A ratio of unity can be assumed to indicate equal rates of cellsurvival and death, as would be expected in the mature kidney at rest,illustrated in FIG. 9. Non-ischemic control kidneys displayed aproliferation:apoptosis ratio of 0.86±0.1, close to the value of unity.Animals pre-treated with saline alone (100 volume of diluent) displayeda significant decrease in the proliferation:apoptosis ratio(0.34%±0.05), indicating that cell death is the predominant feature atthe 24 hour reflow time-point. In contrast, NGAL-treated kidneysdisplayed a marked increase in the ratio of proliferating versusapoptotic tubule cells. This was most evident in animals pre-treatedwith NGAL (2.9%±0.5), but was also evident when the NGAL wasadministered during (2.0%±0.1) or even one hour after (1.7%±0.1) theischemic injury. This analysis indicates that NGAL tilts the overallbalance of tubule cell fate towards cell survival following ischemicinjury.

Example 8

Expression of NGAL increases in Acute Renal Failure of the Human. Acuterenal failure in humans was marked by log order elevations in theconcentration of serum and urinary NGAL protein, shown in FIG. 11.Urinary NGAL protein was 22 ng/ml (n=10) in normal subjects, and 557ng/ml (25-fold elevation, p<0.001) in subjects with acute renal failureand a variety of co-morbidities. Urinary NGAL immunoblots are shown inFIG. 11A, and as quantitative graphs in FIG. 11C. Compared to normalsubjects, in which serum NGAL was 21 ng/ml (geometric mean; n=5)subjects with acute renal failure and a variety of co-morbidities had7.3-fold elevations in serum NGAL (146 ng/ml, p<0.05). These data areshown as immunoblots in FIG. 11B, and as quantitative graphs in FIG.11D. Patients with acute renal failure associated with bacterialinfection tended to have the highest levels of serum (331 ng/ml) andurinary (2786 ng/ml) NGAL, but this was not statistically different fromacute renal failure without infection. To determine whether NGALexpression correlated with the extent of acute renal impairment, we usedsimple regression analysis after log transformation of NGAL levels. Wefound both serum (r=0.64, n=32) and urinary NGAL levels (r=0.68, n=38),as well as urine NGAL normalized for urine creatinine (r=0.67, n=36)were highly correlated with serum creatinine levels (p<0.0001). Incomparison, patients with chronic renal failure had less prominentelevations in serum NGAL (49 ng/ml, n=10) and urine NGAL (119 ng/ml,n=9), and these values failed to correlate with serum creatinine. Thesedata correlate NGAL expression with acute kidney damage, implicating thekidney as the major source of serum and urinary NGAL. Indeed, in severalcases of severe renal failure, the fractional excretion of NGAL (theclearance of NGAL, normalized for the clearance of creatinine) wasgreater than 100%, demonstrating that urinary NGAL derived from localsynthesis, rather than only by filtration from the blood. By comparison,mouse urine also contained markedly elevated levels of NGAL, shown inFIG. 11E, following induction of ATN injury.

To visualize sites of expression of NGAL in acute renal diseases, humankidney tissue sections were stained with affinity-purified polyclonalantibody to NGAL (FIG. 12). The normal kidney demonstrated very weakstaining in the distal tubular epithelia (mean 10% of cortical area) andin medullary collecting ducts, shown at low power in FIG. 12A, and athigh power in FIGS. 12B, and 12C. Rare focal staining of glomerularparietal epithelial cells, but not other glomerular cells was alsoidentified. Proximal tubules however were entirely negative. Incontrast, nearly 50% of cortical tubules were stained for NGAL inkidneys exposed to nephrotoxins or ischemia in kidney sections fromsubjects with the following diagnoses: FIG. 12D, ischemic ATN caused bysepsis; FIG. 12E, hypovolemia (acute loss of blood volume); FIG. 12F,heart failure; FIG. 12G, nephrotoxicity due to bisphosphonate; FIG. 12H,nephrotoxicity due to cephosphorin; and FIG. 12I, hemoglobinuria. NGALwas also widely expressed in the proximal tubule of patients withproliferative glomerulopathies, shown in FIGS. 12J and 12K, but to alower degree than that found in ischemic damage (percentage of corticalparenchyma positive for NGAL was 20% in minimal change disease, 40% indiabetic nephropathy and 50 and 65% in ANCA and anti-glomerular basementmembrane diseases). Tubular cells displaying features of cell injury,including simplification and enlarged reparative nuclei with prominentnucleoli, had the most intense staining. Tubular cells with lessderangement had much less staining. These data demonstrate de novo andwidespread NGAL reactivity in cortical tubules of different renaldiseases and demonstrate that NGAL expression is a common response ofdamaged epithelia in human kidney.

Example 9

Exogenous NGAL Rescues the Mouse Proximal Tubule from ATN. To examinethe functional significance of NGAL expression in renal ischemia, ATNinjury was induced in mice. The renal artery was clamped for 30 min andthe contralateral kidney was removed. Twenty-four hours afterreperfusion, the plasma creatinine rose from 0.41±0.1 mg/dl (n=4) to3.16±0.17 mg/dl (n=8; p<0.001) and NGAL mRNA message and protein wereintensely expressed. NGAL mRNA levels rose approximately 1000 fold,reducing the threshold for detection by Real-Time PCR from 17.7±0.87cycles in sham kidneys to 7.52±0.44 cycles (p<0.0001, n=4 each) inischemic kidneys (normalized to beta actin mRNA levels). NGAL proteinrose 1000 fold in the urine (40 μg/ml in ATN compared to 40 ng/ml in thesham operated and normal mouse, as shown in FIG. 11E), 300 fold in theblood (30 μg/ml in ATN compared to 100 ng/ml in the sham-operated mouse)and was elevated close to 100 fold in kidney extracts (Average 73 μg/gcompared to <1 μg/g kidney wet weight in sham-operated kidney, n-3,p<0.05). The amount of NGAL protein in the kidney correlated well withthe duration of cross-clamping.

To determine whether NGAL was protective in the ischemic model of ATN,we introduced NGAL systemically (1-300 μg by subcutaneous orintraperitoneal injection) prior to, or within one hour of the releaseof the arterial clamp. Injection of μg 100 NGAL 15 minutes beforeclamping blocked the rise in plasma creatinine measured 24 hours afterreperfusion (1.18±0.18 mg/dl, n=7; compared to 3.16±0.17 mg/dl inuntreated animals). Similar data were obtained for dosages ranging from10-300 μg of NGAL, but 1 μg NGAL was not protective(creatinine=3.09±0.11 mg/dl, n=3). Introduction of NGAL one hour afterreperfusion also blocked the azotemia (creatinine=1.60±0.28, n=3,p<0.001), but to a lesser degree than pre-treatment with NGAL. Thesedata were confirmed by measurement of the blood urea nitrogen (data notshown).

The activity of NGAL was also demonstrated by histological findings thatrather than necrotic tubules and luminal debris, normal epithelialmorphology was preserved in the S1 and S2 segments of the proximaltubule, shown in FIG. 13A, in Control and NGAL-treated kidneys(ATN+NGAL), compared to ATN kidneys. The S3 segment in the outer stripeof the outer medulla was less protected by injection of NGAL, buttubular casts were less evident, shown in FIG. 13B. These observationswere supported by Jablonski scoring of the sections, shown in FIG. 13C.In contrast, treatment with NGAL 2 hours after ischemia had noprotective effect (creatinine=3.12±0.35 mg/dl, n=3).

Example 10

Correlates of Ischemia Perfusion Injury. Because the trafficking andmetabolism of the cadherins is rapidly affected by ischemia, and becauseNGAL acts as an inducer of E-cadherin in rat embryonic metanephricmesenchyme, NGAL rescues cadherin expression in the ischemic kidney. Totest this hypothesis we first confirmed that while E-cadherin could bedetected in mouse proximal tubules by immunofluorescence, N-cadherin waspresent in all segments of the proximal tubule, shown in FIG. 14A, andappeared to be its major cadherin. N-cadherin is known to be processedby caspases, β-secretase and by matrix metalloproteinases which generate30-40 Kd cytoplasmic fragments which are potentially important signalingmolecules that modulate CREB signaling. N-cadherin was degraded to a 30Kd fragment after ischemia reperfusion, demonstrated by immunoblot inFIG. 14B, suggesting the activation of one or more of these pathways. Insome animals degradation of the protein could be detected within 6 hoursof reperfusion, and by 24 hours both N-cadherin immunofluorescence andthe full-length protein was nearly abolished. In contrast, pre-treatmentwith NGAL preserved N-cadherin immunofluorescence, enhanced theexpression of full length N-cadherin and reduced the appearance of itsfragment when monitored at 6 hours (in some animals) or 24 hours ofreperfusion. Hence, the preservation of proximal tubule markerN-cadherin correlates with and is a sensitive marker of NGAL activity.E-cadherin, which is highly expressed in the distal tubule andcollecting duct, was much less affected by ischemia and by NGALtreatment. Similarly, metal induced nephrotoxic ATN triggered thedegradation of N-cadherin but not E-cadherin. One effect of NGAL,therefore, is to inhibit signaling by N-cadherin fragments.

Because disruption of the proximal cell results in apoptotic cell death,the effect of NGAL on cell viability was determined using a TUNEL assayof apoptosis induction. Twenty-four hours after reperfusion, we countedthe percentage of tubules with at least one TUNEL-positive tubular cell,shown in FIG. 14C. Ischemic kidneys (I/R) showed 11.5%+0.6 (SEM, n=4animals) of cortical tubules contained TUNEL-positive cells, but aftertreatment with NGAL (I/R+Ngal), the percentage of positive tubules fellto 2.9%±0.9 (SEM, n=7; p<0.001). By comparison, 0.5%±0.3 of corticaltubules had TUNEL-positive cells in sham kidneys, shown in thequantitative graph in FIG. 14D.

BrDU uptake was determined as a method to measure cell proliferation bydetermining the percent of cortical tubules with at least oneBrDU-positive tubular cell in histological sections of kidneys (notshown). Ischemic cortical tubules contained rare BrDU-positive cells(1.9%+0.3; n=3) while ischemic kidneys pretreated with NGAL had a smallbut significant increase in positive cells (3.9%±0.5; n=4; p<0.05)measured 24 hours after the insult. By comparison, 3.7%±0.7 of corticaltubules had BrdU-positive cells in sham kidneys. Hence, rescue by NGALreduced apoptosis of cortical cells and either stimulated compensatorytubular cell proliferation or rescued cells from damage.

Because the expression of NGAL correlates with ischemic damage,endogenous NGAL expression after treatment with exogenous NGAL proteinwas measured. Treatment of ischemic animals with 100 mg NGAL reduced theincrease in endogenous NGAL RNA by 72%±16 (p<0.01; n=5) at 24 hours ofreperfusion as measured by real time PCR. Treatment with 100 mg NGALreduced the appearance of endogenous NGAL protein in the kidney by 2.5fold (ischemia 73±7 μg/g; NGAL-treated ischemia, 29±7 μg/g; n=3 each;p<0.01) as measured by immunoblot, preserved tubular cells withproliferation potential.

Example 11

NGAL Upregulates Heme Oxygenase-1 in ATN. A number of studies haveidentified heme oxygenase 1 (HO-1) as a critical regulator of theproximal tubule in renal ischemia. HO-1 is necessary for recovery fromATN and its level of expression is directly correlated with the rescueof tissue damage. As shown in FIG. 14E, ischemia reperfusion (ATN lanes)enhanced the expression of HO-1, but when ATN-injured mice were treatedwith 10-100 μg NGAL (ATN Ngal lanes), the enzyme was further upregulated5-10 fold by 24 hours after reperfusion. To determine whether NGAL aloneinduced HO-1, healthy mice (Sham lanes) were injected with increasingdoses of NGAL, and HO-1 protein levels were measured. However, theexpression of HO-1 after NGAL injection was much less than theNGAL-treated ATN-injured kidneys, indicating that NGAL synergizes withother activators to upregulate HO-1 during ischemic-reperfusion eventsand protect the kidney from iron-mediated damage.

Example 12

Mechanism of Rescue from ATN: NGAL Targets the Proximal Tubule.Distribution of exogenous NGAL was determined after an intraperitonealor subcutaneous injection to establish the mechanism by which NGALprotects the proximal tubule from ischemic damage. NGAL was found in theurine within 10 min of injection of 100 μg exogenous NGAL suggestingthat the protein was rapidly cleared by the kidney, shown in FIG. 15.The same time course was observed following injection of 10 μg NGAL (notshown). However, only 0.1-0.2% of the injected NGAL was recovered in theurine in the first hour. To better follow trafficking, fluorescentconjugates of NGAL were administered. Both fluorescein- andAlexa-labeled NGAL localized to large vesicles in the subapical domainof the cortical proximal tubule (S1 and S2 segments of the nephron) byone hour, but not to other segments of the tubule, shown in FIG. 16A. Itshould be noted that protein trafficking itself is also unlikely to bethe mechanism of renal protection because a second lipocalin, retinoidloaded RBP, which is also captured by the proximal tubule and degradedin lysosomes was ineffective (FIG. 6A). To determine if these organelleswere lysosomes, we labeled proximal tubular lysosomes withfluorescein-dextran (43 kDa) the day before administering Alexa-568NGAL. One hour after injecting NGAL, 33% of the NGAL vesicles alsocontained dextran, shown in FIG. 16B. In addition, many of thesevesicles co-stained with the lysosomal marker LAMP1 (data not shown).Similar results were observed following injection of [¹²⁵I]-NGAL, FIG.16C, which showed that the full length protein was rapidly cleared fromthe blood and located in the kidney by the one hour time-point. In fact,the kidney had 13-fold more [¹²⁵I]-NGAL than the liver/mg protein.Nearly identical data were previously reported with human NGAL, whichrapidly cleared the circulation (t_(1/2)=10 minutes) and located in thekidney at levels 12-fold higher than the liver/mg protein. Thekidney-localized protein was TCA precipitable (70%), and composed ofboth full-length NGAL and a specific 14 kDa degradation product. Thesespecies persisted, and were only slowly lost after 5 hours afterinjection. In contrast, the plasma, and particularly the urine containedmostly low molecular weight, TCA soluble [¹²⁵I]-fragments, (35% and 20%TCA precipitable, respectively).

These data show that full length NGAL is rapidly cleared by the proximaltubule where it traffics to lysosomes and degrades to a 14 kDa fragment.It is likely that the endogenous protein (low levels of serum NGAL)traffics in a similar manner, because there is very little urinary NGALin normal mouse or human urine, despite the fact that it is freelyfiltered from the circulation (human: filtered load=20 ng/ml X GFR,whereas urine NGAL=22 ng/ml; mouse: filtered load=100 ng/ml X GFR,whereas urine NGAL=40 ng/ml).

Example 13

Rescue of the Proximal Tubule from ATN Requires Fe:Siderophore. Todetermine if NGAL can deliver iron to the proximal tubule, NGAL wassaturated with the radionuclide iron species ⁵⁵Fe by incubatingiron-free enterochelin-NGAL (Sid:NGAL) with ⁵⁵Fe at a 1:1 stoichiometry(55Fe:Sid:NGAL). One hour after injecting this radiolabeled⁵⁵Fe:Sid:NGAL complex (10 μg intraperitoneal), the majority of ⁵⁵Fe wasrecovered in the kidney (55%), while only trace amounts were found inthe plasma (4.3%), urine (0.6%), liver (2.4%), and spleen (0.2%). Todetermine the location of the ⁵⁵Fe in the kidney, radioautography oftissue sections was performed. ⁵⁵Fe was localized in the proximaltubule, particularly along the apical surface, beneath the brush border,as shown in FIG. 16E, and in Table 1, where X²=21.2 and p=0.0017.

TABLE 1 SilverGrains^(a) Area^(b) Relative Location (% Total) (% PointCount) SA^(c) X² Lumen 26.46 18.49 1.43 3.43 Apical MB 12.91 5.84 2.218.54 Cytosol 48.98 48.81 1 0.00062 Nucleus 4.02 7.17 0.56 1.39 Basal MB3.81 6.41 0.59 1.06 Interstitium 3.71 12.2 0.3 5.91 Glomerular Tuft 0.131.16 0.11 0.91 Key: ^(a)Total silver grains = 2601 ^(b)Total point count= 999 ^(c)% grains/% point count.

In contrast, ⁵⁵Fe was not found in the medulla, shown in FIG. 16D. Thesedata demonstrate that both the NGAL protein and its ligand, iron, can becaptured by the proximal tubule when exogenous ⁵⁵Fe:Sid:NGAL complex isinjected. It should be noted that the distribution of ⁵⁵Fe:Sid:NGAL wasquite different from the distribution of non-protein bound ⁵⁵Fe citrate,wherein only 1.5% of the iron was recovered in the kidney (not shown).

Example 14

To determine the role of iron delivery in renal protection, we comparedNGAL cloned in two different strains of E. coli bacteria. NGAL cloned inXL-1Blue bacteria contains enterochelin and is iron-loaded, while NGALcloned in BL-21 bacteria does not contain enterochelin. Ten μg of XL-1Blue-cloned NGAL (holo-Ngal 10 μg, left panel) protected the kidney incomparison with sham-treated (Sham) and untreated ATN-injured kidney(ATN), shown in FIG. 17A. However, there was a reduced level ofprotection with 10 μg of BL-21-cloned NGAL (apo-Ngal 10 μg, centerpanel) lacking enterochelin. Therefore, BL-21-cloned NGAL wasreconstituted with iron-free (apo-Ngal:Sid) and iron-saturatedenterochelin (apo-Ngal:Sid:Fe). Loading with enterochelin (with orwithout iron) enhanced the protection of the kidney and blunted the risein serum creatinine. The presence of iron was slightly less protective,since iron-saturated siderophore has a diminished capacity for chelatingiron in the kidney. Because both the iron-loaded form and the ironfree-form of NGAL:Sid were protective, it is possible that thesiderophore itself, rather than iron was active. To test the role ofiron further, a gallium:Sid:NGAL:Sid:gallium (apo-Ngal:Sid:Ga) complexwas also tested. Because gallium is a metal that occupies iron bindingsites with high affinity, including the enterochelin siderophore(gallium blocks ⁵⁵Fe binding to enterochelin to the same extent asunlabeled iron), and because it can not undergo redox reactions typicalof iron, gallium competes with iron for binding to the siderophore. Incontrast to the iron complex, mice treated with the gallium complex 15minutes prior to ischemia were not protected (creatinine=3.17±0.1; n=4).In addition, a single dose of free Siderophore (Sid), desferrioxaminemesylate (DFO) or retinol-binding protein (RBP) failed to protectagainst ATN. These data demonstrate that NGAL:siderophore complexesprovide the protective activity of NGAL, and that iron transport by thesiderophore is dependent upon NGAL. An immunoblot of NGAL:Siderophorecomplexes with or without iron (Fe) are shown in FIG. 17B.

Example 15

Demonstration of a Urine Siderophore. The actions of endogenous NGAL invivo might differ from the pharmacological effect of exogenous NGAL,because the critical siderophore associated with exogenous NGAL is abacterial product. The presence of endogenous low molecular weightfactors that transport iron, however, have been suggested by a varietyof studies. These molecules can include citrate, and related compounds,but also iron-transporting activities that have a molecular weight inthe range of 1 Kd. To determine whether an NGAL co-factor is present inthe urine, apo-NGAL from BL21 bacteria was mixed with urine samples fromhealthy mice. While the low molecular weight components of the urine (<3Kd) failed to trap ⁵⁵Fe above a 10 Kd cut-off filter, and apo-NGALdiluted in Tris or phosphate buffer failed to trap ⁵⁵Fe, incubation ofNGAL with urine (<3,000 Da) permitted the retention of ⁵⁵Fe, shown inFIG. 18A. The capture of iron by NGAL was inhibited by the presence of1000-fold unlabeled iron citrate, and more powerfully by a 50-foldconcentration of iron-saturated enterochelin, shown in FIG. 18B. Thecapture of iron was saturable, and when using 30 μl of mouse urine,approximately 20% of NGAL molecules bound iron. These findings suggestthat mouse urine contains a low molecular weight co-factor that permitsNGAL-iron interactions. Because the endogenous factor is competitivewith the bacterial siderophore, which binds the calyx with high affinity(0.4 nM), it appears that both bacterial and mammalian factors occupythe same binding pocket of the lipocalin.

Example 16

NGAL expression in patients undergoing kidney transplantation. Humansundergoing kidney transplantation were evaluated to determine NGALexpression during the recovery period. Kidney biopsies were obtainedwithin 1 hour of transplantation from living related donors (LRD, n=10)or cadaveric (CAD, n=12) kidney transplants. Biopsy specimens weresectioned and immunohistochemically stained with NGAL antibody. NGALexpression was significantly increased in the CAD group, as shown inFIG. 19. Since cadaveric kidneys are maintained outside the body for alonger period of time than is typical for kidneys from living relateddonors, the degree of ischemic injury is generally greater, and waspositively correlated by NGAL expression.

Western blots of urine samples obtained prior to transplantation, andwithin 2 hours of transplantation from LRD (n=4) or CAD (n=4) kidneytransplants, shown in FIG. 20. NGAL expression in the urine was absentbefore the operation. NGAL expression was significantly increased in theCAD group compared to the LRD group. Quantitation of urinary NGALmeasured by Western blots in LRD versus CAD showed a significantlyincreased expression in CAD, shown in FIG. 21. Quantitation by ELISAdemonstrated similar results (not shown). This finding again correlatedwith the longer period of ischemia associated with CAD kidneytransplantation. Correlation of urinary NGAL obtained 2 hours after CADtransplantation with cold ischemia time, shown in FIG. 22.

The serum creatinine levels, which peaked at 2-4 days after thetransplant surgery occurred, also correlate with the urinary NGAL.Correlation of urinary NGAL obtained 2 hours after CAD transplantationwith peak serum creatinine is shown in FIG. 23.

Example 17

Use of NGAL measurement as a diagnostic tool for Acute Renal Failure.One of the unfortunate outcomes of cardiopulmonary bypass (CPB) duringopen heart surgery is the development of acute renal failure (ARF).Serum NGAL measurement can be highly predictive for patients who at riskof developing ARF. Standard curves for NGAL ELISA are shown in FIG. 24,the linear relationships obtained from 10 independent standard curves.Serial serum NGAL was measured in samples from patients who developedARF following CPB (n=10), shown in FIG. 25. NGAL was markedly elevatedin samples collected after surgery, and remained elevated for at least 4days. In contrast, patients who did not develop ARF had no increases inserum NGAL levels during the first 4 post-operative days. FIG. 26 shownmeans ±SD for serial serum NGAL in patients who developed ARF (n=10)versus those who had an uneventful postoperative course (n=30) Serialurine NGAL measurements in patients who developed ARF following CPB(n=11) was also elevated as shown in FIG. 27, but was more variable thanserum NGAL. However, urine NGAL levels were predictive of ARF, as shownin FIG. 28, with and analysis of means±SD for serial urine NGAL inpatients who developed ARF (n=11) versus those who had an uneventfulpostoperative course (n=30). Urine NGAL levels at 2 hr postoperativecorrelated with length of CBP time during surgery, shown in FIG. 29.

While the present invention has been illustrated by the description ofembodiments and examples thereof, and while the embodiments and exampleshave been described in considerable detail, it is not intended torestrict or in any way limit the scope of the appended claims to suchdetail. Additional advantages and modifications will be readily apparentto those skilled in the art. The invention in its broader aspects istherefore not limited to the specific details, representative methodsand structures, and illustrated examples shown and described.Accordingly, departures can be made from such details without departingfrom the scope or spirit of the invention.

1. A method of treating, reducing, or ameliorating a toxin-induced renalinjury in a mammal, comprising the step of administering exogenousneutrophil gelatinase-associated lipocalin (NGAL) in an amount effectiveto treat, reduce or ameliorate the toxin-induced renal injury.
 2. Themethod according to claim 1, wherein the NGAL is administeredintravenously or parenterally.
 3. The method according to claim 1,wherein the toxin-induced renal injury results from a condition,treatment, or therapy selected from the group consisting of contrastagent treatment, antibody treatment, and antibiotic treatment.
 4. Themethod according to claim 1, further comprising the step ofadministering to the patient a siderophore in an amount effective toenhance the treating, reducing, or ameliorating of the toxin-inducedrenal injury effected by the exogenously administered NGAL.
 5. Themethod according to claim 4, wherein NGAL and the siderophore areco-administered as a complexed compound of NGAL and the siderophore. 6.The method according to claim 1 wherein the step of administeringexogenous NGAL comprises administering a composition comprising NGAL, inan amount effective to treat, reduce, or ameliorate the toxin-inducedrenal injury.
 7. The method according to claim 6, wherein thecomposition comprises at least 10 microgram NGAL per 100 microliter ofcomposition.
 8. The method according to claim 7, wherein the compositioncomprises at least 100 microgram NGAL per 100 microliter of composition.9. The method according to claim 6, wherein the composition furthercomprises a siderophore.
 10. The method according to claim 9 wherein theNGAL and siderophore are a complexed compound.
 11. The method accordingto claim 1 wherein the NGAL is administered in an amount from about 1 toabout 200 mg NGAL per kg body weight of the mammal.
 12. The methodaccording to claim 1 wherein the toxin-induced renal injury results froma toxin selected from the group consisting of an antibiotic, ananti-inflammatory agent, an antifungal agents, a radio-contrast agent, apharmaceutical, a chemotherapeutic agent, a test drug, a medicamentsubstance, and naturally-occurring, commercial and industrial chemicalsand minerals.
 13. The method according to claim 12 wherein the inducingtoxin is selected from the group consisting of a cancer chemotherapeuticincluding cisplatin, mitomycin, cyclophosphamide, isosfamide, andmethotrexate, an antibiotic including gentamicin, vancomycin, andtobramycin, an antifungal agent including amphotericin, ananti-inflammatory agent including an NSAID, an immunosuppressantincluding a cyclosporine and tacrolimus, a chemical including ahydrocarbon, a chlorocarbon and a fluorocarbon, a mineral includingarsenic, mercury, bismuth and lead, an aminoglycoside, foscarnet,pentamidine, vancomycin, neomycin, nitrous oxide, isoflurane, kanamycin,and cyclophosphamide.
 14. The method according to claim 1 wherein thetoxin-induced renal injury results from administering a toxin selectedfrom the group consisting of an antibiotic, an anti-inflammatory agent,an antifungal agents, a radio-contrast agent, a pharmaceutical, achemotherapeutic agent, a test drug, a medicament substance, andnaturally-occurring, commercial and industrial chemicals and minerals.15. The method according to claim 14 wherein the administered toxin isselected from the group consisting of a cancer chemotherapeuticincluding cisplatin, mitomycin, cyclophosphamide, isosfamide, andmethotrexate, an antibiotic including gentamicin, vancomycin, andtobramycin, an antifungal agent including amphotericin, ananti-inflammatory agent including an NSAID, an immunosuppressantincluding a cyclosporine and tacrolimus, a chemical including ahydrocarbon, a chlorocarbon and a fluorocarbon, a mineral includingarsenic, mercury, bismuth and lead, an aminoglycoside, foscarnet,pentamidine, vancomycin, neomycin, nitrous oxide, isoflurane, kanamycin,and cyclophosphamide.
 16. The method according to claim 14 wherein theexogenous NGAL is administered prior to, during or at the same time as,or following, the administering of the toxin.
 17. The method accordingto claim 16 wherein the exogenous NGAL is administered during or at thesame time as the administering of the toxin.
 18. The method according toclaim 16 wherein the exogenous NGAL is administered prior to theadministering of the toxin.
 19. A method of treating, reducing, orameliorating an injury selected from an ischemic injury, anischemic-reperfusion injury, and a toxin-induced injury, to an organ ina patient, the organ selected from the group consisting of a liver,heart, brain, lung, stomach, intestine, colon, pancreas, blood vessels,bladder, cervix, skin, and portions thereof, comprising the step ofadministering to the patent neutrophil gelatinase-associated lipocalin(NGAL) in an amount effective to treat, reduce, or ameliorate the injuryto the organ.
 20. A method of preventing an injury selected from anischemic injury, an ischemic-reperfusion injury, and a toxin-inducedinjury, to an organ in a patient, the organ selected from the groupconsisting of a kidney, liver, heart, brain, lung, stomach, intestine,colon, pancreas, blood vessels, bladder, cervix, skin, and portionsthereof, comprising the step of administering to the patent neutrophilgelatinase-associated lipocalin (NGAL) in an amount effective to preventthe injury to the organ.